Introduction: 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.
Objective: 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. Methods: 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.
Results: 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.
Conclusions: 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.
Bibliographical noteFunding Information:
This work was supported in part by the UK Natural Environment Research Council (NE/P010326/1) and Thermo Fisher Scientific, via iCASE PhD studentship to JMM.
We thank Gavin Lloyd and Andris Jankevics (Phenome Centre Birmingham, UK), Elena Sostare (Michabo Health Science, UK) and Emilio Gonzalez Oses (Beckman Coulter) for their scientific advice. We thank Martin R. Jones (The Swiss Federal Institute of Aquatic Science and Technology, Switzerland) for his scientific advice and reviewing the manuscript. We thank Dr Christiane Guguen-Guillouzo, Dr Philippe Gripon, Dr Christian Trepo for their development work of the HepaRG cell line.
© 2022, The Author(s).
- Chemical risk assessment
- Direct infusion mass spectrometry
- High-throughput screening
- In vitro metabolomics
ASJC Scopus subject areas
- Endocrinology, Diabetes and Metabolism
- Clinical Biochemistry