Physiological Oxygen is Healthier for Cell Cultures

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Physiological Oxygen is Healthier for Cell Cultures

Drs. Timpano and Uniacke, Hypoxystation users at University of Guelph in Ontario, have published a very thorough study examining the molecular basis of cells’ reactions to differing levels of hypoxia. In their paper “Human Cells Cultured Under Physiological Oxygen Utilize Two Cap-binding Proteins to Recruit Distinct mRNAs for Translation” (Journal of Biological Chemistry 291:20; 2016), they examine 2 different translation initiation proteins, eiF4E and eiF4E2, that are activated under either high (>8% O2) or low (<1% O2) oxygen levels, with the aid of mTORC1 or HIF-2α, respectively, and activated simultaneously in an area of low- to mid-level physioxia (1-8% O2). Timpano and Uniacke were able to stably and accurately create low oxygen in their Hypoxystation by Hypoxygen, which provides a closed workstation environment that enables researchers to culture and manipulate cells inside the chamber through gloveless sleeves, eliminating the negative consequences of spikes of higher oxygen and lower temperatures encountered in an incubator as cell cultures are growing. Their research into translational modulation of the proteome using the Hypoxystation gives seminal insights into physioxia as the natural condition for cells, both in vitro and in vivo.

“Culturing cells in ambient air could be far from physiological with resepct to oxygen. Oxygen is a surprisingly neglected factor (in cell culture)” – Dr Timpano and Dr Uniacke, University of Guelph, Ontario, Canada

Through polysome association experiments with cells growing at ambient air versus lower oxygen levels of 1%, 3%, 5%, and 8%, RNA analysis, and m7-GTP cap-binding assays, Timpano and Uniacke were able to demonstrate that the oxygen concentration in the workstation was sufficient to either repress or increase the activity of eiF4E and eiF4E2, reflecting mechanisms that occur during development but also during tumor progression and in ischemic diseases. Cells can reversibly cycle between utilisation of the eiF4E protein, which preferentially binds to the 5′ TOP mRNA’s at >8% O2 and is impaired at hypoxia, and eiF4E2, which is active at <1% O2 and utilizes binding motifs in the 3′ UTR of the mRNA. The eIF4E type of mRNA’s code for housekeeping proteins while the eIF4E2-dependant mRNA’s encode signaling proteins needed to respond to environmental signals, allowing cells to control translation dynamically and giving cancer cells an edge during tumor progression, as hypoxia increases.

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