Abstract
Most cell culture is performed under atmospheric oxygen (20.9%), but this level is not present in vivo. Cells cultivated under more physiologic O2 levels have been reported to exhibit different responses, but O2 levels in the culture media have not been reported in such studies. We utilized a SensorDish Reader® to monitor real time O2 levels in the culture media of human umbilical vein endothelial cells (HUVEC) exposed to 170 μM (20.9%), 30 μM (3%), and 10 μM (1%) O2 concentrations for up to 24 hrs. Approximately 6 hrs were required for the media of unstirred cultures to achieve the final level of O2. By western blot analysis, hypoxia inducible factor 1 alpha (HIF‐1A) was not detectable at 170 μM O2 but was readily detectable at 30 and 10 μM. At the end of 24 h RNA was isolated and subjected to gene expression analysis with human whole genome microarrays. About 740 genes were statistically altered in their expression with a false discovery rate <5%. The gene most altered in its expression by reduced O2 was EGLN3, a key enzyme in regulating HIF‐1A levels. The growth of HUVEC over 6 days did not differ at 170 μM and 30 μM, but experienced a 20% increase in time to confluence at 10 μM O2 indicating that the level of oxygen was limiting for growth. Some discrepancies reported between behavior of freshly isolated cells versus cells cultured for only a few days in vitro might be explained by their growth in hyperoxic conditions.
Published Version
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