OP42 - Precision control of dissolved oxygen in mammalian cell culture media impacts on in situ volatile generation and promotes improved mesenchymal stem cell yield accompanied by reduced transcriptional variability

Abstract

Listen

Translate article

Mammalian cell culture is reliant on culture media to support in vitro growth and proliferation. Culture media contains salts, vitamins, and minerals at precise levels where subtle variations result in novel media formulations. Oxygen is a bioactive molecule which impacts strongly on intracellular signalling, mitochondrial function, and metabolic pathway choices. We sought to determine the levels of dissolved oxygen in cell culture media and to design systems to support precision control of those levels. Dissolved oxygen levels in culture media displayed substantial variability ranging from 6 – 11 mg/L (~10% O2 - 21% O2) in all medias tested (DMEM, MEM, IMDM). In addition to this variability it was apparent that deoxygenation of cell culture media required incubation in hypoxic (1% O2) environments for >24 hours. The deoxygenation process could be accelerated to 3 hours via a combination of cooling (4 °C), agitation, and provision of a hypoxic headspace (1% O2, 5% CO2). Deoxygenation rates were volume dependent but were significantly more rapid than passive approaches. Selected Ion Flow Tube (SIFT)- MS) was then used to measure changes in volatile organic compound (VOCs) in media incubated overnight in either 21% O2, 2% O2, and 2% O2 following rapid deoxygenation. Reduced variation was apparent in pre-deoxygenated media where reduced standard deviations revealed significant differences to base media in 40% of VOCs tested. Finally we sought to determine the impact of pre-deoxygenated media on clonogenicity and transcriptome of MSCs isolated from fresh marrow. Precision deoxygenation (2% O2) of culture media associated significantly with increased yields of MSCs. Microarray analysis of MSCs isolated in all conditions further demonstrated reduced variability in global transcriptome where differentially expressed transcripts associated strongly with defined pathways. In summary precision control of dissolved O2 levels reduces media-based chemical reaction variability and promotes enhanced cell isolation from primary materials.