What Oxygen Level Does My Cell Culture Actually Experience?


Read about 3 protocol factors that affect your cell cultures below.

Well, it depends upon your cell culture protocol.

Just like in the perfect cookie recipe1, there are many factors in cell culture that affect your results. There is one set of ingredients or reagents that we don’t often think about when placing cultures into the incubator and shutting the door: the gasses. Even the 5% CO2 reading on the incubator isn’t what your cells are actually experiencing; not until the cell cultures have equilibrated with the incubator.

That takes time: time that your cells are out of optimum conditions.

Likewise, with an oxygen-controlled incubation chamber, it takes time for cell culture to equilibrate with the incubator oxygen levels. There are multiple levels that have to come to equilibrium first. There is the gas phase outside of the vessel, the headspace within the vessel, the gas/medium interface, and the medium at the pericellular level.

At each of these levels, different factors, determined by your protocol, change the exchange rate and the time that your cells are out of optimum. Let’s break it down.

Protocol Factors: Gas exchange between the incubator and the vessel headspace

Factors affecting the gas exchange between the incubator and the headspace in the cell culture vessel include the efficiency of air circulation in the incubator and the type and the size of the vessel. Unlike flasks, plates and dishes provide gas exchange gaps around all sides of the vessel. If your protocol uses a flask, there are factors like the tightness of the cap’s seal and the presence of a filter in the cap.

The cell culture vessel you choose for your protocol can change oxygen exchange time between the chamber and the incubation space.


Protocol Factors: Gas exchange between the headspace and the cell culture medium

Oxygen dissolves quite poorly2 into static aqueous solutions from gas phase. Between the headspace and the medium liquid, factors such as whether the culture is static or stirred make a tremendous difference. Also, the surface area for gas exchange and the starting gas levels of the medium contribute to the time to come to equilibrium. Is your static culture flask lying flat, tipped at an angle, or standing on end? These are protocol factors that affect oxygen equilibration between the headspace and the medium.

Protocol Factors: Gas exchange between the medium surface and the pericellular level

Factors get far more variable once the conditions at the bottom of the flask or plate are considered, especially in static (unstirred) cultures. Live cell cultures are never static themselves, and living things, by definition, continuously modify their environment. It is patently obvious to cell culturists that pericellular conditions change over time. Without even looking at the cells under a microscope, we can see the color change of cultures grown with phenol red dye and see the opacity of the medium change as the cultures grow denser.

Oxygen equilibrates poorly in aqueous solutions. In static culture, cells absorb oxygen from the medium, creating a gradient of oxygen between the pericellular level and the medium surface. As cultures grow, the overall oxygen demand from one day to the next also grows. The distance between the medium surface and the cells also has an effect on the equilibration time.

So cell density, culture age, and medium depth are factors in your protocol that affect pericellular oxygen levels for your cells. Oxygen consumption rates of a single culture are variable, even in steady log-phase growth. Keep cells in a consistent cell density range from experiment to experiment to limit undesirable variability.

Some manufacturers offer oxygen-permeable cell culture vessels that allow oxygen exchange through the bottom of the vessel in static culture. This eliminates the headspace exchange time and reduces the diffusion distance between the chamber and your cells, reducing the time for equilibration.

At every level, the beginning gas levels on each side of the exchange affect the time to come to equilibration. Change your protocol. Pre-equilibrate your medium to the desired oxygen level and avoid mixing the medium with room air in the BSC to reduce the time your cells are out of optimum.

Summary for protocol factors affecting your cell cultures:

Pericellular oxygen levels depend upon multi-stage gas exchange between the chamber and the cells in the culture vessel.

Factors in your protocol for static culture that affect oxygen diffusion to your cells include vessel type and medium depth.

Cell density affects oxygen consumption rate which affects pericellular oxygen levels. Use consistent cell density ranges from experiment to experiment and don’t let cells overgrow.

Some vendors sell culture vessels with oxygen-permeable growth surfaces.

Add to your protocol the step of pre-equilibration of your medium to the desired gas content, in order to reduce the time out of optimum for your cells.

1www.ozy.com/good-sht. The science behind baking the most delicious cookie ever. Anne Miller. May 20, 2015.

2 www.pftforum.com/blog. Gas solubility and why it matters. Richard Johnston. May 26, 2014.


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.