Cell culture under oxygen controlled conditions: an improvement towards more predictive results

Oxygen concentration in tissues is a key factor for cell and organ survival. In normal conditions, partial oxygen pressure (pO2) results in the balance between oxygen delivery and its consumption. Oxygen is transported, in mammals, by circulating red blood cells. Partial oxygen pressure in tissues varies widely, depending on their respective metabolic requirements and their functional status. In normal physiological conditions,  partial oxygen pressure is called physioxia. Any alteration of tissue environment leading to a decrease in partial oxygen pressure is called hypoxia. Hypoxic conditions have been observed in many different pathological situations like tumor development, obesity or transcient ischemia.Sensory neurons

In vitro cell cultures are widely used for fundamental research and their behaviour are extensively analysed in response to various stress, treatments or changes in their environment. The possibility to perform assays on cells or cell lines derived from specific organs increases predictivity of in vivo tests and reduces animal testing. In different domains such as cosmetics, decisions made to avoid animal testing increase pressure on in vitro models to guarantee both compound efficiency and absence of side effects.
The most common cell culture techniques use plated or suspension cells in 95% air – 5% CO2 controlled incubators. These are called normoxic conditions, as oxygen is provided by breathing air concentration. Almost all in vitro studies have been performed in these normoxic conditions and results regularly expected to be predictive to what happens in vivo. But oxygen concentration in the air is around 21%, which means in a 95% air – 5 % CO2 controlled incubator, final oxygen concentration is 20%.

Table 1 : Normal values of pO2 in various tissues, expressed in percentage of oxygen in the microenvironment (1)

Table 1 : Normal values of pO2 in various tissues, expressed in percentage of oxygen in the microenvironment (1)

Compared to O2 concentrations in different normal tissues (click on Table 1 to enlarge), usual cell culture conditions appear to be abnormally hyperoxic.

What are the effects at different oxygen concentrations?

Modulation of oxygen concentration has been described to have a direct effect on cell behaviour. Many hypoxic experiments have demonstrated induction of the well known Hypoxic induced factor (HIF). But it’s not the only factor involved. For example, microRNAs (miRNAs) which are short (20-24 nucleotide) non-coding RNAs that negatively regulate gene expression, are also impacted when oxygen environment is altered. Unexpectedly, even cell adhesion molecule expression has also been described to to be modulated by oxygen modification (2).
A recent poster analysed normal human adipocytes from different body mass indicators (BMI) to differentially express protein and miRNA biomarkers when maintained in hypoxic conditions, which is the normal adipocyte environment in case of obesity. Oxygen modulation induces expression of various biomarkers (3).

Performing cell cultures with a 21% oxygen concentration has consequences on physiological responses observed. Results may suffer some bias compared to in vivo conditions. An interesting example is the assay for anti-oxydant properties of active compounds. Isn’t there a huge risk of false negative results as cell are already in huge oxydative stress? Mainly the most active – and probably the most toxic – compounds will appear efficient. If experiments were conducted at 6-8% O2, there is a reasonable chance to detect other active compounds and with fewer side effects.

For what kind of projects?

Oxygen tension in cell culture is not the exclusivity of tumor cell projects and the oncology environment. The increasing need to identify relevant biomarkers for diagnostic purposes, as well as for companion products, emphasizes the importance of initially well built protocols, in order to identify the most relevant and predicitive biomarkers. Within this strategy, in vitro studies performed in the appropriate oxygen environment are among the primary factors to take care of.

tebu-bio offers various services, among which cell cultures performed under strictly controlled conditions are available. Access to a Hypoxy-Cool device that generates a determined oxygen concentration in bottled media offers a unique versatile capacity to handle quick oxygen variations in cultured cells. This allows to reproduce specific pathological situations where oxygen bursts or deprivation are observed.

With cell cultures performed alone or in combination with their biomarker identification platform, tebu-bio offers convenient access to highly sophisticated tools for scientists to develop unique and more predictive projects.


(1) Carreau A., El Hafny-Rahbi B., Matjuk A., Grillon C. and Kieda C. “Why is partial oxygen pressure of human tissues a crucial parameter? Small molecules and hypoxia” , J. Cell. Mol. Med. 2011; vol. 15; pp. 1239-1253
(2) Kulshreshtha R., Ferracin M., Wojcik SE., et al. « A microRNA signature of hypoxia » Mol. Cell. Biol. 2007; vol. 27; pp. 1859-1867
(3) Mennesson, E., Fixe I., Foucher A., Carpentier F. and Normand N. « miRNA and protein biomarkers in hypoxia for obesity studies », Biomarker meeting, Munich, November 2014