More Tips for Extreme Oxygen Studies:

Anoxia, Hypoxia, Physioxia and Hyperphysioxia

Cytocentric Basics: What oxygen level do the cells in my lab actually experience?

Researchers often shut the door on an incubator or chamber, check that the numbers on the outside for CO2 and O2 look good, and then turn away, assured that their cells are at the proper gas levels. However, setting up the atmosphere in any chamber does not mean that your cells inside experience that oxygen level.

Gases have to equilibrate with the liquid medium before they get to cells. Because oxygen does not dissolve well into aqueous solutions, if the medium is not pre-equilibrated to the desired oxygen levels, cells will experience unintended oxygen levels for a long time until the medium equilibrates with the chamber. See more information on that in a previous post.


Hypoxia, Physioxia, and Pericellular Oxygen

If cells are consuming oxygen in the medium, they will drive the dissolved oxygen lower than the gas atmosphere in the vessel headspace [1]. So if you equilibrate the medium to an atmosphere at 1-2% O2, the pericellular oxygen may rapidly fall to 0% O2 with cells in the mix. How fast it falls depends upon factors like the cell density, cell type, cell metabolic state, the volume of medium, whether it is static or agitated, and type of cell culture vessel. So the conditions your cells experience are quite protocol-dependent.

A 2015 review by Wenger, et al is a terrific resource for considering all the factors that go into pericellular oxygen levels[2]. In this paper, the authors explain in detail why room air incubators aren’t at 21% oxygen, as most researchers state in the methods sections of their papers. They also explore what you might find if you investigated the oxygen levels that your media might contain.


Anoxia: Use 0.1% instead of 0.0% Oxygen

If you set an oxygen controller to 0.0% oxygen, computers being literal, it will use up all or most of your tanked N2 getting to and maintaining 0.00% O2. Oxygen sensors, in general, have a margin of error of about 1% at the very low end of their range. This means that 0.1% O2, is effectively 0% O2.

So, because 0.1% isn’t detectably different from 0%, plus the fact that any cells in the medium will rapidly consume what little oxygen is there, and the probability that the controller will run your tank out of nitrogen overnight trying to get to absolute zero, 0.1% O2, as a setting, produces a more reliable 0% than 0.0%.


Hyperphysioxia: Don’t Set Controllers to 95% Oxygen in a Humidified Chamber

Just as everyone that reports that their room air incubator is at 21% oxygen is wrong, everyone reporting 95%O2 and 5%CO2 in humidified chambers is wrong. While a gas tank may contain 95%O2/5%CO2, those gases in the tank are dry. Water vapor takes up a significant amount of gas volume in the presence of an open water source; about 8% of the total volume of a chamber at 37ºC. If you use a programmable chamber to try to bring O2 in a humidified chamber up to 95%, while maintaining 5%CO2, you’ll never get there. As Wenger et al discusses, how close you get depends upon your altitude, but one customer reported a maximum of 87% oxygen in a humidified chamber with 5% CO2. It pays to go beyond just hooking up a known tank of gas and actually check what the cell culture media are exposed to in the chamber.


Measure the Pericellular Dissolved Oxygen

The only way to determine the gas levels that your cells are actually experiencing is to measure it. Buy a dissolved oxygen sensor and check the cell culture medium inside the vessel both near the air interface and near the cells. It may change your approach to your experiments.


So I Found This Old Oxygen Chamber and Controller in the Lab (or Somebody Else’s Lab)

It would be great to just dust it off, hook everything up and get started, but there may be a few other things to consider. Most importantly, how long ago was this apparatus purchased?

The oxygen sensor may need to be replaced if it is more than 2 years old. Oxygen sensors continually “burn” once they are exposed to air, so that oxygen sensor that has been sitting around for awhile may need replacing even if it hasn’t been in use. Also, with time and air exposure, the adhesives that hold magnetic door seals in place may degrade.

No matter the age of your system, the most important step is to calibrate the instrument before you start experiments. Then you can be sure that you have the most accurate readings for your experiments.


Contact BioSpherix Service for any questions. We are happy to help.


1. 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.

2. Wenger, R.H., et al., Frequently asked questions in hypoxia research. Hypoxia, 2015. 3: p. 35-43.