Three Reasons Not to Use 21% Oxygen In Vitro

You can finally control the gases around your cell cultures chambers and controllers and are all ready to jump into experiments, but where do you start? One study people often report is the comparison of physiologically appropriate oxygen levels with 21% oxygen.

Here are three reasons why you should not use 21% oxygen in vitro.

Your Room Air Incubator Isn’t at 21% Oxygen

If your goal is to make a comparison with your old conditions, setting a chamber to 21% oxygen won’t do that. The oxygen level for uncontrolled air, 20.9%, is the standard for outdoor air that is completely dry at sea level. No water vapor at all. This, of course, isn’t the condition inside your incubator or chamber, which has an open water source for very high humidity.

Water vapor takes up space inside the incubator and its relative contribution increases with increasing altitude. This isn’t because the partial pressure of water vapor above the liquid is changing, but because the mass of the other gases decreases [1].

Room air incubators not only have added water vapor, but also added CO2. We have measured the oxygen levels inside standard COincubators, after opening the door just long enough to place a sensor on a shelf.  After the CO2 charged back up inside, we found an average of about 17% oxygen. If the door stayed closed, the oxygen levels drifted closer to 19% over hours to days. This was in different institutions at altitudes between 300 and 500 feet above sea level.

Opening and closing the incubator door, however, made the oxygen levels spike higher as room air entered, and then drop sharply lower again as the COrecharged. We could drive oxygen levels all over the place just by opening and closing the incubator door.

So 21% oxygen isn’t a level that your cells were exposed to in your old incubator. They were far more likely to have been cultured in a highly variable environment in a range between about 17 and 19% oxygen. If you want to compare your new controlled conditions with your old variable and uncontrolled conditions, don’t use 21% oxygen in a chamber, just keep some cultures in your old incubator.

Room Air Oxygen is Irrelevant for Physiologic Studies.

Where in the body do you find live cells exposed to room air oxygen? Maybe at the nasal epithelium? The oral cavity?

The cells at the body surfaces exposed to room air tend to be dead and/or coated in mucus or fluid. As soon as air enters the body in the airways, it immediately mixes with expired gases, diluting room air oxygen. By the time it gets to the lungs, oxygen is down from 21% to 10-13% oxygen.

Inside the body, each tissue type has an oxygen range that is far below room air oxygen. It is important to choose an appropriate range for your cell type of model. One useful discussion of in vivo oxygen ranges, although it is older, is found in Carreau et al[2].

 It is difficult to get good numbers on the oxygen levels found in vivo. Oxygen is never one number, but found in gradients depending upon local blood circulation and physiology. One group reported an oxygen gradient in the outer layers of skin that drops off sharply as an oxygen-sensing microprobe penetrated from outer to inner layers [3]. Of course, any oxygen-sensing method that disturbs the tissues may alter the circulation there, but this paper and others give us a sense of the types of gradients found in outer tissues.

So while 21% is a nice number for room air, it really doesn’t have any place in physiologically relevant studies.

While Important, the Chamber Atmosphere Isn’t the Most Important Factor

The pericellular oxygen, the oxygen level the cells actually experience, can also vary considerably from the chamber oxygen[4]. Chamber gases have to diffuse into the vessel headspace, then dissolve in the medium, then diffuse to the cells sitting on the bottom or the vessel. Because oxygen is extremely slow to dissolve in aqueous solutions, it is important to pre-equilibrate medium and other fluids that contact your cells or your cells will be out of optimum for hours while waiting for their oxygen levels to equilibrate with the chamber.

Another way to reduce the time to pericellular oxygen equilibration is to use cell culture vessels that are oxygen permeable. That way the cells are sitting on the surface that exchanges gases with the chamber.

Measuring the pericellular oxygen level in your in vitro system can provide you more relevant data. It might also lead to protocol changes that can help get your cells closer to the physiological conditions that you want to test.


  1. 1. Wenger, R.H., et al., Frequently asked questions in hypoxia research. Hypoxia, 2015. 3: p. 35-43.
  2. 2. Carreau, A., et al., Why is the partial oxygen pressure of human tissues a crucial parameter? Small molecules and hypoxia. J Cell Mol Med, 2011. 15(6): p. 1239-53.
  3. 3. Wang, W., C.P. Winlove, and C.C. Michel, Oxygen partial pressure in outer layers of skin of human finger nail folds. J Physiol, 2003. 549(Pt 3): p. 855-63.
  4. 4. Bambrick, L., Y. Kostov, and G. Rao, In vitro cell culture pO2 is significantly different from incubator pO2. Biotechnology progress, 2011. 27(4): p. 1185-1189.


alicia author iconAbout the Author

Alicia D Henn, PhD, MBA

Alicia Henn has been the Chief Scientific Officer of BioSpherix, Ltd for two years. Previously, she was a researcher at the Center for Biodefense Immune Modeling in Rochester, NY. Alicia obtained her PhD in molecular pharmacology and cancer therapeutics from Roswell Park Cancer Institute in Buffalo, NY and her MBA from the Simon School at University of Rochester in Rochester, NY.