An engineered culture vessel and flow system to improve thein vitroanalysis of volatile organic compounds

Abstract

AbstractVolatile organic compounds (VOCs) are a biologically important subset of an organism’s metabolome, yetin vitrotechniques for the analysis of these small molecules vary substantially in practice, restricting the interpretation and reproducibility of study findings. Here, we present an engineered culture tool, termed the “Biodome”, designed to enhance analyte sensitivity by integrating dynamic headspace sampling methodology for the recovery of VOCs from viable biological cultures. We validate the functionality of the device forin vitrovolatile metabolomics utilizing computational modeling and fluorescent imaging of mammalian cell culture. We then leverage comprehensive two-dimensional gas chromatography coupled with a time-of-flight mass spectrometer and the enhanced sampling capabilities afforded by our tool to identify seven VOCs not found in the media or exogenously derived from the sampling method (typical pitfalls within vitrovolatilome analysis). We further work to validate the endogenous production of these VOCs using two independent approaches: (i) glycolysis-mediated stable isotopic labeling techniques using13C6–D-glucose and (ii) RNA interference (RNAi) to selectively knockdown β-oxidation via silencing ofCPT2. Isotope labeling reveals 2-Decen-1-ol as endogenously derived with glucose as a carbon source and, through RNAi, we find evidence supporting endogenous production of 2-ethyl-1-hexene, dodecyl acrylate, tridecanoic acid methyl ester and a low abundance alkene (C17) with molecular backbones likely derived from fatty acid degradation. To demonstrate applicability beyond mammalian cell culture, we assess the production of VOCs throughout the log and stationary phases of growth in ampicillin-resistant DH5αEscherichia coli. We identified nine compounds with results supporting endogenous production, six of which were not previously associated withE. coli. Our findings emphasize the improved capabilities of the Biodome forin vitrovolatile metabolomics and provide a platform for the standardization of methodology.