Versatile set-up for non-invasive in vitro analysis of headspace VOCs.

Volatile organic compound (VOC) profiles emitted in trace concentrations from bacteria or cells has gained increasing importance over the decades. Analysis of VOCs in the headspace does not interfere with in vitro systems and, therefore, offers new options for non-invasive monitoring of cultures. Currently there is not any available standardized in vitro sampling system which considers effects of dilution and contamination onto ppbV to pptV VOC concentrations during. In this study a new in vitro system for online and offline headspace measurement of biological cultures was designed. The system was built from inert materials, equipped with universal sampling ports and easily adjustable volume options. Standard VOC mixtures in the system were analyzed by means of proton-transfer-reaction time-of-flight mass spectrometry and needle-trap-microextraction coupled with gas chromatography/mass spectrometry with a variance of 5%-14% and 10%-15%, respectively. In a proof of concept setup volatile emissions over cell cultures and pure media were assessed. The newly developed system enabled reliable and reproducible headspace analyses of in vitro cultures. As parallel application of different analytical methods is possible and confounding factors could be minimized, this set-up represents an important step towards standardization of headspace analysis over biological cultures.

Smell of cells: Volatile profiling of stem- and non-stem cell proliferation.

Bacterial and cell cultures are known to emit a large number of volatile organic compounds (VOCs). Conventional biochemical methods are often destructive, time-consuming and expensive. In contrast, VOC analysis of headspace over cultures may offer a non-destructive alternative for the monitoring of cell proliferation and metabolism. VOC profiles from cultures of murine pluripotent stem cells and fibroblasts were assessed every 24 h for 3 days. Pure cell media were measured as parallel controls. VOC analysis was highly standardized with respect to time of measurement and phases of cell growth. Cultures were grown in custom-made inert boxes. In order to determine the effects of fresh media supply on VOC emissions, both cell types were cultured with and without daily media exchange. VOCs from headspace were preconcentrated by means of needle trap micro-extraction and analysed by gas chromatography-mass spectrometry (GC-MS). Murine pluripotent stem cells emitted increasing concentrations of thiirane and methyl-methoxy-hydroxy-methyl-amine (MMHA). Substance concentration correlated with cell numbers. Murine fibroblasts did not emit thiirane or MMHA. Concentrations of aldehydes, especially benzaldehyde, were lower in both cell cultures than in pure media samples. Daily media exchange resulted in higher cell numbers, but had no major effects on VOC concentrations emitted from the cells. Investigation and monitoring of volatile substances such as thiirane and MMHA may enhance the understanding of stem cell properties and lead to a destruction-free characterization of pluripotent stem cells.

VOC breath profile in spontaneously breathing awake swine during Influenza A infection.

Influenza is one of the most common causes of virus diseases worldwide. Virus detection requires determination of Influenza RNA in the upper respiratory tract. Efficient screening is not possible in this way. Analysis of volatile organic compounds (VOCs) in breath holds promise for non-invasive and fast monitoring of disease progression. Breath VOC profiles of 14 (3 controls and 11 infected animals) swine were repeatedly analyzed during a complete infection cycle of Influenza A under high safety conditions. Breath VOCs were pre-concentrated by means of needle trap micro-extraction and analysed by gas chromatography mass spectrometry before infection, during virus presence in the nasal cavity, and after recovery. Six VOCs could be related to disease progression: acetaldehyde, propanal, n-propyl acetate, methyl methacrylate, styrene and 1,1-dipropoxypropane. As early as on day four after inoculation, when animals were tested positive for Influenza A, differentiation between control and infected animals was possible. VOC based information on virus infection could enable early detection of Influenza A. As VOC analysis is completely non-invasive it has potential for large scale screening purposes. In a perspective, breath analysis may offer a novel tool for Influenza monitoring in human medicine, animal health control or border protection.