Abstract
Regulatory bodies have started to recognise the value of in vitro screening and metabolomics as two types of new approach methodologies (NAMs) for chemical risk assessments, yet few high-throughput in vitro toxicometabolomics studies have been reported. A significant challenge is to implement automated sample preparation of the low biomass samples typically used for in vitro screening. Building on previous work, we have developed, characterised and demonstrated an automated sample preparation and analysis workflow for in vitro metabolomics of HepaRG cells in 96-well microplates using a Biomek i7 Hybrid Workstation (Beckman Coulter) and Orbitrap Elite (Thermo Scientific) high-resolution nanoelectrospray direct infusion mass spectrometry (nESI-DIMS), across polar metabolites and lipids. The experimental conditions evaluated included the day of metabolite extraction, order of extraction of samples in 96-well microplates, position of the 96-well microplate on the instrument’s deck and well location within a microplate. By using the median relative standard deviation (mRSD (%)) of spectral features, we have demonstrated good repeatability of the workflow (final mRSD < 30%) with a low percentage of features outside the threshold applied for statistical analysis. To improve the quality of the automated workflow further, small method modifications were made and then applied to a large cohort study (4860 sample infusions across three nESI-DIMS assays), which confirmed very high repeatability of the whole workflow from cell culturing to metabolite measurements, whilst providing a significant improvement in sample throughput. It is envisioned that the automated in vitro metabolomics workflow will help to advance the application of metabolomics (as a part of NAMs) in chemical safety, primarily as an approach for high throughput screening and prioritisation.
Highlights
New approach methodologies (NAMs) are understood as alternative methods to vertebrate animal testing that provide greater robustness, throughput and/or mechanistic knowledge into risk assessment, thereby enabling more relevant decision making for human health and the environment
The workflow presented in this study offers an automated approach for in vitro metabolomics for application in NAMs to advance the pace of chemical risk assessment
We have developed, characterised and demonstrated an automated sample preparation and analysis workflow for in vitro metabolomics in 96-well microplates, using a Biomek i7 Hybrid Workstation (Beckman Coulter) and spectral-stitching nESI-DIMS (Thermo Scientific Orbitrap Elite) for polar metabolites and lipids
Summary
New approach methodologies (NAMs) are understood as alternative methods to vertebrate animal testing that provide greater robustness, throughput and/or mechanistic knowledge into risk assessment, thereby enabling more relevant decision making for human health and the environment. Some examples of NAMs include in vitro, in silico, and in chemico approaches, high-throughput screening (HTS), omics technologies or combinations thereof [1,2,3]. Omics technologies are capable of providing comprehensive information on the molecular response of a biological system to external stimuli such as exposure to a chemical. Metabolomics provides the most downstream molecular measurements of the omics technologies, enabling a phenotypic readout of a biological system at a particular time [9]. Similar to high-throughput transcriptomics (HTTr), applying metabolomics in combination with HTS (i.e., large-scale assays employing automation) could provide a tool for rapid identification and characterisation of responses of biological systems to chemical exposure [11], yet would require increased throughput of metabolite extractions
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