Rapid and effective modulation of dissolved oxygen levels in cell culture media for improved hypoxia studies
Cell-based hypoxia studies are reliant on close control of O2 concentration in the surrounding environment. Exposure to ambient O2 (21%) concentrations for short or sustained periods of time can result In confounding results due to oxygen toxicity. Reduced Oz cell culture incubators and workstations are examples of adopted measures designed to reduce this oxygen toxicity and to promote consistency of observation. We have identified a further layer of complexity to this scenario - culture media. With a starting 02 concentration of approximately 10% 02, a high salt content, and high operating temperature (37-C) it can take culture media approximately 3 hrs to reduce to 2% 02 in the presence of cells and essentially never In the absence of cells and in large volumes.
To overcome this deficiency we have developed a system for the rapid and effective modulation of dissolved oxygen levels in cell culture media. We have engineered a simple agitation device within a refrigerated unit with both 02 and C02 gas level control. The system will reproducibly deoxygenate 500 ml bottled volumes of culture media with sterility retained through vented cap incorporation. Deoxygenation of large medium volumes to defined set-points (0.5%, 1%, 2%, etc) is readily achievable within 90 minutes. Controlled experimentation to evaluate human mesenchymal stem cell (hMSC) recovery from bone marrow aspirate in a 2% 02 workstation revealed a 50% improvement in both colony-forming unit-fibroblasts (CFU-F) and in absolute cell numbers when pre-deoxygenated (2% 02) medium was used as compared to medium stored in 2% 02 workstation prior to its use. The dramatic improvement in cell number was accompanied by widespread global transcriptional alterations at both transcript and exon levels as identified through Exon LOST microarray analysis. This included 167 and 71 unique upregulated and down-regulated transcripts as compared to 2% 02 workstation-alone recovered hMSC when compared to 21% 02 recovered hMSC. In summary we have developed a system for the rapid and effective modulation of dissolved oxygen levels in cell culture media. The application of this technology resulted in improved hMSC CFU-F and cell number recovery which was accompanied by widespread global transcriptional changes.