When Should You Culture Cells at 21% Oxygen?
Technical Note
Never
The only cells that experience 21% atmospheric oxygen are those on the outermost layer of the skin and the cells in the trachea and bronchioles entering the lungs. All the other cells of the body under physiological conditions experience oxygen levels significantly lower than 21%. Typically, between 1-8% is the physioxic range for most internal organs. Therefore, the best models for true understanding of cell biology should never culture cells at 21% oxygen.
This figure summarizes oxygen levels in human tissues.
Historic, Not Scientific
For over a century, tissue culture has been performed under what is considered normoxic (21% oxygen) conditions. However, the reason a vast majority of peer reviewed publications describe using 21% oxygen is not scientific but only historic. There is no scientific evidence that justifies performing these experiments at atmospheric oxygen levels. In fact, extensive evidence supports the statement that cells cultured under appropriate physiological conditions express in vivo regulator genes that are silent at normoxic concentrations (e.g. HIF, Hypoxia Inducible Factor). Malaria parasites, lymphocytes, embryos and stem cells to name a few, all require hypoxic environments for optimal cell function.
It is only in the latter half of the 20th century that a few researchers designed in vitro experiments reflecting physiological levels of oxygen in situ. That is, in vitro environments that reflect in vivo conditions, (i.e. hypoxic environments, 0.1-10% O2).
Adapted illustration from NIH BIOART Source.
Turn Down Oxygen to Turn Up Research Reproducibility
The ideal path forward is to reduce oxygen levels for all cell culture applications from basic research to applied cellular therapeutics. Adoption needs to be wide and universal to accelerate successful research and cell-based therapies. While the lack of reproducibility in research studies has focused on cell line authenticity, reagent quality, and poor lab practices, among many others, the control of oxygen levels and cell culture conditions has been overlooked. If we seek to truly solve research irreproducibility, the known variability in using artificial oxygen levels at 21% needs to end. Learn how Embrient’s low-cost solutions simplify effective physioxic environments.
References
- Carreau A, El Hafny-Rahbi B, Matejuk A, Grillon C, Kieda C. Why is the partial oxygen pressure of human tissues a crucial parameter? Small molecules and hypoxia. J Cell Mol Med. 2011 Jun;15(6):1239-53. doi: 10.1111/j.1582-4934.2011.01258.x.
- Alva R, Gardner GL, Liang P, Stuart JA. Supraphysiological Oxygen Levels in Mammalian Cell Culture: Current State and Future Perspectives. Cells. 2022 Oct 4;11(19):3123. doi: 10.3390/cells11193123.
- Taylor CT, Scholz CC. The effect of HIF on metabolism and immunity. Nat Rev Nephrol. 2022 Sep;18(9):573-587. doi: 10.1038/s41581-022-00587-8. Epub 2022
Jun 20.
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