What Oxygen Level Should I Use for My Cells In Vitro?

Well, it depends.

Learning how to determine oxygen levels for your in vitro culture is important to producing relevant results.

Are you looking for physiological relevance or comparison with cells cultured in a standard room-air incubator?

First, you should know that there is nothing normal or “normoxic” about room air oxygen for cells that normally reside inside the body.In vivo, as soon as air enters the body, it mixes with CO2 being expired and the percentage of oxygen decreases.


In vitro, while an incubation chamber can certainly be controlled to 20.8% oxygen (generally accepted to be room air), this is supraphysiologic.

If you want to assay your cells in comparison with a standard CO2-controlled incubator, you also should not use 20.8% oxygen. We’ve measured the oxygen in those incubators and it is lower, 17.2 +/- 0.3% (n=4 incubators in three different institutions) when it first equilibrated. This value slowly drifted to 19% if the door was kept closed for three days.

Why are incubator oxygen levels lower than room air?

The CO2 is set to 5% and room air is usually less than 1% CO2. Every time you open the inner door, CO2 is infused to replace the CO2 lost to the room and it displaces oxygen. Also, high humidity means that water vapor necessarily takes up gas volume in your incubator; up to 8%. See relevant data in a previous post.

I want physiologically-relevant oxygen for my cells. How much should I use?

Oxygen levels in the body vary widely depending upon the physiologic or pathophysiologic context. Venous blood is generally accepted to contain 5% oxygen. Some tissues, like bone marrow or fetal tissues, can normally experience much lower oxygen levels. Tissues with little vasculature, like cartilage or the interior of large solid tumors, can be close to anoxic (0%).

We strongly recommend that you refer to scientific literature related to your specific cell or tissue model to determine the best oxygen level for your cell culture.

These reviews can help you get started:

1. Jež M, Rožman P, Ivanović Z, Bas T. Concise Review: The Role of Oxygen in Hematopoietic Stem Cell Physiology. Journal of cellular physiology. 2015;230(9):1999-2005.

2. Carreau A, Hafny‐Rahbi BE, Matejuk A, Grillon C, Kieda C. Why is the partial oxygen pressure of human tissues a crucial parameter? Small molecules and hypoxia. Journal of cellular and molecular medicine. 2011;15(6):1239-53.

3. Mohyeldin A, Garzon-Muvdi T, Quinones-Hinojosa A. Oxygen in stem cell biology: a critical component of the stem cell niche. Cell stem cell. 2010;7(2):150-61.


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.