Most cell culture is still done in atmospheric oxygen, but that’s not the environment cells actually experience in the body. Experts say controlling oxygen can make in vitro systems more physiologically relevant, with important implications for everything from stem cell work to cancer research. But before labs invest in specialized equipment, it’s worth understanding the difference between normoxia, physioxia, and hypoxia—and when oxygen control really matters.
In this Ask the Expert article from Biocompare, Zachary Rogers, Postdoctoral Fellow in the lab of J. Christopher Love at the Koch Institute for Integrative Cancer Research at MIT, shares his perspective on why oxygen control is becoming an increasingly important consideration in cell culture.
Biocompare: What is oxygen-controlled cell culture? What is the difference between hypoxic and physioxic cell culture?
Zach: “Conventional cell culture is performed in ambient air, 18.6% O2 after taking into consideration humidity and 5% CO2. This is higher than cells experience in the body—0.5% in the intestinal lumen and up to 13% O2 in the lungs. Oxygen-controlled cell culture attempts to overcome this limitation and provide cells with a specific oxygen tension. Physioxic cell culture is performed at oxygen concentrations experienced in the body (in practice usually 5% O2) and hypoxic cell culture includes any oxygen tension that triggers the stabilization of hypoxia-inducible factors, or HIFs (typically 1% O2).”
Biocompare: How does one perform this method? What commercial options are available?
Zach: “Oxygen-controlled cell culture products operate under the same principle—cells are placed inside a chamber in which the oxygen concentration is manipulated by adding compressed nitrogen. This includes portable chambers, which are placed inside conventional cell culture incubators, tri-gas incubators, incubators with nitrogen, and workstations, which are combined biological safety cabinets and incubators for controlled oxygen cell culture handling and incubation.”
Biocompare: What are some of the use cases for oxygen-controlled cell culture?
Zach: “Culturing cells in supraphysiological oxygen concentrations reduces the Hayflick limit, increases reactive oxygen species exposure, and alters metabolism. Oxygen-controlled cell culture has been used to model tissues in physioxia, including bone marrow, adipose, brain, liver, etc. Oxygen control is important for many stem cell expansion and differentiation protocols. Hypoxic cell culture is useful for studying how cells sense low oxygen (e.g., HIF pathway) and cancer metastasis.”
Biocompare: What are some of the considerations when designing oxygen-controlled cell culture experiments?
Zach: “Although oxygen-controlled cell culture equipment controls the gas phase surrounding cells, this does not necessarily mean the pericellular oxygen tension, or the oxygen that cells experience is well-controlled. This is mainly because cellular oxygen consumption is non-negligible, particularly at lower oxygen concentrations in the gas phase. The cell culture vessel, media volume, oxygen concentration, cell density, and cell type all play a role in the pericellular oxygen tension. Therefore, it is important to consider the pericellular oxygen tension when performing these experiments. Pericellular oxygen tension can even drop significantly in conventional cell culture.”
Biocompare: Should all cell culture be performed under oxygen control?
Zach: “Although controlled oxygen improves the ability of cell culture to model tissues in the body, it is a specific type of experiment that requires specific equipment and consideration. One challenge is that oxygen-controlled cultures are rapidly reoxygenated when removed from their respective incubators. The implications are not fully understood, but it is clear that reoxygenation destabilizes HIFs, generates reactive oxygen species, and can confound results. The workstations avoid this issue by allowing for cell culture handling and passaging in an oxygen-controlled environment. Lastly, cells will not necessarily survive in hypoxic conditions for long periods. Therefore, oxygen control can be useful when it is pertinent to the experimental design but should not necessarily replace conventional cell culture.”
Biocompare: What practical factors should labs consider before investing in oxygen-controlled cell culture equipment?
Zach: “Depending on the number and depth of oxygen-controlled experiments, there are available products for all types of these experiments. Portable chambers are low cost and easy to implement in a cell culture workflow. Tri-gas incubators require capital investment but are straight-forward to use. Workstations are the gold standard but are expensive. When deciding to pursue these types of experiments, it’s important to consider what oxygen concentration is needed, how reoxygenation will be handled, and the relationship between the gas phase and pericellular oxygen tension for a given set-up.”