Volatile biomarkers emitted by cell cultures: headspace analysis using selected ion flow tube mass spectrometry, SIFT-MS

Abstract

The work presented in this thesis was initiated in order to develop a non-invasive real-time gas phase analytical technique, based on selected ion flow tube mass spectrometry (SIFT-MS), for monitoring the progression of human cell cultures and the detection of microbial contamination in such cultures by monitoring and quantifying the emitted volatile compounds.

Fundamental SIFT experiments were performed to characterise the reactions of the SIFT-MS precursor ions (H3O+, NO+ and O2 +?) with several volatile compounds of potential value to biological research; a necessity for their quantification. The work has resulted in new methods for the quantification acetaldehyde and CO2 in gaseous samples.

The compounds present in the headspace of sealed cultures of six human cell types were analysed by SIFT-MS, the key finding being their consumption of the toxic volatile compound acetaldehyde from the media. Further experiments involved the addition of the enzyme aldehyde dehydrogenase inhibitors diethylaminobenzaldehyde and disulfiram to cultures of hepG2 (hepatocellular carcinoma) cells, when it was observed that consumption of acetaldehyde from the cultures/headspace was reduced, and in some cases, acetaldehyde was even produced due to the actions of the cellular alcohol dehydrogenase enzyme. Furthermore, the solvent dimethylsulphoxide was reduced to imethylsulphide by the cells, which is known to occur via the enzyme methionine sulphoxide reductase. This process was retarded by the ALDH inhibitors.

The use of SIFT-MS for the detection of microbial infection in mammalian cell cultures was also explored. The volatile compounds emitted by E. coli (strain JM109), into the gas phase above two different culture media, were analysed using SIFT-MS. Further, the progression of a culture of this bacterium was monitored continuously over a 4-hour period. The findings of this research were then applied to the study of human cell cultures intentionally infected by E. coli bacterium, including cultures contained in a 1L bioreactor.