Is Oxygen Sabotaging Your Research?

Technical Note

The evidence has been mounting, supported by thousands of studies, demonstrating the problems with cell culture performed in 21% oxygen (normoxic). Mammalian cells under physiological conditions in vivo typically exist in homeostatic conditions with oxygen levels between 1-8%, known as physioxia. Thus, the common practice of culturing cells at 21% oxygen can lead to artificial results that do not reflect true biology.


Normoxic oxygen concentrations either suppress entirely or limit the expression of many genes and cellular pathways in culture. Results of experiments carried out in this kind of abnormal environment may be misleading. Check our many citations for proof. Nobel Prize-winning studies clearly show that cells cultured under appropriate hypoxic physiological conditions, express critical genes in vivo, such as HIF-1 that are silent at higher, normoxic concentrations. Gene regulation, cellular proliferation, differentiation and viability are all sensitive to oxygen concentrations. Furthermore, cellular metabolic pathways are significantly altered between physioxic and normoxic states. These effects are observed across a wide array of virtually all cell types, including but not limited to stem cells, immune cells, neuronal cells, embryos, muscle cells, and cancer cells, as well as infectious agents such as malaria. The implications on cell culture are vast, affecting not only routine cell line propagation, but also immune cell isolation and activation, hybridoma development and growth, drug candidate screening and testing, and cell cultures for adoptive cell therapies.


  • The discrepancy between in vivo and in vitro oxygen status can have detrimental effects on experiment interpretation.
  • Most stem cells and neuronal cells thrive in hypoxic conditions.
  • Exposure to lower, physiological oxygen conditions increases survival, proliferation and differentiation.
  • Some stimuli are toxic to cultured cells in atmospheric oxygen but effectively stimulate in physiologically relevant oxygen conditions.
  • Given the known consequences of oxidative stress on cells, it only makes sense to perform experiments in a physiological environment.
  • Research may be reproducible but relevant interpretation of results may be impossible due to inappropriate oxygen conditions.
  • Compound toxicity testing on cancer cells that thrive under hypoxic conditions may be inaccurate at high oxygen levels.




Our Modular Incubator Chamber provides an airtight environment for the creation of hypoxic, hyperoxic or
normoxic conditions. Learn more about the many benefits of the incubator chamber.



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