Integrating in vitro metabolomics with a 96-well high-throughput screening platform.
Integrating in vitro metabolomics with a 96-well high-throughput screening platform.
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DOI：
10.1007/s11306-021-01867-3
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发表时间：
2022-01-09
期刊：
影响因子：
--
通讯作者：
Viant MR
中科院分区：
文献类型：
--
作者：
Malinowska JM;Palosaari T;Sund J;Carpi D;Bouhifd M;Weber RJM;Whelan M;Viant MR
高通量筛选(HTS)正在成为支持化学品安全评估决策的一种方法。同时，体外代谢组学是一种很有前途的方法，可以帮助加速在毒性测试中从动物模型向高通量细胞模型的过渡。在这项研究中，我们建立并评估了一种与96孔HTS平台兼容的高通量代谢组学工作流程，每孔使用50,000个HepaRG肝细胞。利用一种新建立的半自动方法提取低生物量细胞样本进行代谢组学分析，并使用高分辨率光谱拼接纳米电喷雾直接进样质谱仪(NESI-DIMS)分析细胞内代谢物。对从细胞培养和采样到代谢表型测量的整个工作流程的灵敏度和重复性进行了评估，显示出足够的灵敏度(肝细胞提取物中的>； 3000特征)和板内和板间重复性(中位数相对标准偏差 <； 为30%)。这些分析被用于以模型毒物氯化镉进行原则证明毒理学研究，揭示了在48小时暴露期间的五个采样时间内代谢物的变化。为了允许进行脂类组学分析，通过建立极性代谢物和脂类的分离提取方法，扩展了溶剂系统。这里报告的实验、分析和信息学工作流程在灵敏度、可重复性和检测毒物引起的代谢组变化的能力方面符合预定义的标准，并已准备好应用于代谢组学驱动的毒性测试，以补充HTS分析。网上版载有补充材料，可在10.1007/s11306-021-01867-3查阅。
High-throughput screening (HTS) is emerging as an approach to support decision-making in chemical safety assessments. In parallel, in vitro metabolomics is a promising approach that can help accelerate the transition from animal models to high-throughput cell-based models in toxicity testing. In this study we establish and evaluate a high-throughput metabolomics workflow that is compatible with a 96-well HTS platform employing 50,000 hepatocytes of HepaRG per well. Low biomass cell samples were extracted for metabolomics analyses using a newly established semi-automated protocol, and the intracellular metabolites were analysed using a high-resolution spectral-stitching nanoelectrospray direct infusion mass spectrometry (nESI-DIMS) method that was modified for low sample biomass. The method was assessed with respect to sensitivity and repeatability of the entire workflow from cell culturing and sampling to measurement of the metabolic phenotype, demonstrating sufficient sensitivity (> 3000 features in hepatocyte extracts) and intra- and inter-plate repeatability for polar nESI-DIMS assays (median relative standard deviation < 30%). The assays were employed for a proof-of-principle toxicological study with a model toxicant, cadmium chloride, revealing changes in the metabolome across five sampling times in the 48-h exposure period. To allow the option for lipidomics analyses, the solvent system was extended by establishing separate extraction methods for polar metabolites and lipids. Experimental, analytical and informatics workflows reported here met pre-defined criteria in terms of sensitivity, repeatability and ability to detect metabolome changes induced by a toxicant and are ready for application in metabolomics-driven toxicity testing to complement HTS assays. The online version contains supplementary material available at 10.1007/s11306-021-01867-3.
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影响因子：
6.1
作者：
Ramirez T;Strigun A;Verlohner A;Huener HA;Peter E;Herold M;Bordag N;Mellert W;Walk T;Spitzer M;Jiang X;Sperber S;Hofmann T;Hartung T;Kamp H;van Ravenzwaay B
通讯作者：
van Ravenzwaay B
影响因子：
6.1
作者：
Chetwynd, Andrew J.;David, Arthur
通讯作者：
David, Arthur
影响因子：
4.2
作者：
Parsons, Helen M.;Ekman, Drew R.;Viant, Mark R.
通讯作者：
Viant, Mark R.
影响因子：
7.4
作者：
Fuhrer, Tobias;Heer, Dominik;Zamboni, Nicola
通讯作者：
Zamboni, Nicola
影响因子：
9.8
作者：
Joossens, Elisabeth;Macko, Peter;Whelan, Maurice
通讯作者：
Whelan, Maurice