bp042 image01What is the O2 Level in My Cell Culture Incubator?

(Hint: It’s Not 21%)

Because room air is assumed to be near 21% oxygen, researchers often report that their room air incubator is as well. However, if you would think about it for a minute, you would realize that this is impossible.

 We charge a traditional open room cell culture incubator with CO, raising it to 5% or more, to maintain the pH of carbonate buffer-based cell culture media. Room air contains only 0.03 - 0.06% CO2 (or sometimes a little more if your ventilation is poor). Every time the inner incubator door is opened and closed, CO2 is infused to replace what is lost to the room. This necessarily drives the percentage of oxygen down inside the incubator.

High humidity also drives incubator oxygen levels down as water vapor can take up about 8% of the gas volume in an incubator with an open water pan. We have had customers with high oxygen applications report to us that they could not drive oxygen levels higher than 87% with 5% COand an open water source.

To measure what oxygen levels actually were in cell culture incubators out in the biomedical wild, we packed up an oxygen sensor and went around measuring oxygen levels in other labs. We opened the inner door of standard room-air 5% CO2-controlled incubators with open water pans just long enough to place a sensor on a shelf before closing the door gently.

We found levels stabilized at an average of 17.2 +/- 0.3% oxygen

(n=4 incubators in 3 different institutions in 2 Upstate NY cities).


We were able to monitor this effect better in the completely controlled setting of an Xvivo barrier isolator containing different sizes of incubators.

bp042 image02We set the processing chamber surrounding the incubators to room air conditions (21% O2 and 0.05%CO2) and the incubators to 21% O2 and 5%CO2 with an open water reservoir. Then we turned off the oxygen control and observed the effect on Olevels in a large (12” x 14” x 22”) incubator when we opened the door, removed a T75 flask and closed the door. We replaced the flask after 20 minutes, simulating normal room air incubator use.

This is what we recorded:

bp042 image03Predictably, opening the door caused CO2 levels to drop. Once closed, the incubator then infused CO2 to restore it to 5% which drove oxygen levels down inside, close to the 17% we had seen in the room air incubators. We shared this data once before in a previous blog post on Reproducibility, but thought it important to share it again here.

The inside volume of the incubator and the frequency of door opening events changed how wide the oxygen swings were. The most volatile conditions we found were with a small incubator (12’ x 14’ x 10”) and more frequent door opening events:

bp042 image04This is what we measured.

As all cells do react to environmental conditions, your results may vary.

It may pay to think carefully what variability you are introducing into your experiment if you do not control oxygen levels for your cells, both in the incubator and during cell handling. Technology does exist that can eliminate these oxygen swings.

Have your own experiences with variable oxygen in the room air incubator? Let us know This email address is being protected from spambots. You need JavaScript enabled to view it.. We would be interested to hear your experiences and thoughts.


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.