Normoxia: Are we cramping cell culture?
During strenuous exercise, anaerobic conditions develop in our muscles as cells rapidly use oxygen. Muscle cells switch to an alternative metabolic pathway that releases lactic acid to cope with reduced oxygen levels. When lactic acid builds up, it can cause cramps. Similarly, media depth can dramatically impact oxygen levels in cell culture. Standardizing this variable, or agitation may help to improve data reproducibility and relevance in cell culture.
Normoxia is the normal level of oxygen that cells experience in living organisms and tissues. In healthy tissue, normoxia is typically 4%-8%, depending on the tissue. So how does this compare to oxygen levels in cell culture? The percentage of oxygen in the air of a cell culture cabinet is 21%. However, as with living organisms, this is higher than the oxygen availability to cells.
Several factors impact the concentration of oxygen available to cells in culture. Firstly, in contrast to liquids, gases are compressible. Consequently, the partial pressures of all the gases in the air of an incubator (including the evaporated water molecules from the water tray) at a given altitude, are additive and must be equal to the atmospheric pressure (Dalton’s law). This law determines the true normal concentration of oxygen in the air of a humidified incubator, which at sea level is 18.6% (Wenger et al 2015).
Secondly, the depth of media will impact oxygen availability. Flick’s first law of gas diffusion states that the diffusion rate of oxygen is proportional to the depth of liquid. Incorporating this law of physics more acutely predicts the concentration of oxygen that a monolayer of adherent cells in culture will experience i.e. the pericellular oxygen concentration.
Finally, the oxygen consumption rate (OCR) of the cells, and the number of cells, will determine the actual level of oxygen at the monolayer.
A new publication from researchers at the Metabolic Research Laboratories in Cambridge and their collaborators Tan et al (2024) utilized miniature oxygen-sensing microprobes to determine the oxygen level above a monolayer of confluent terminally differentiated 3T3-L1 adipocytes. Under their standard cell culture conditions (100 mL of medium/well in a 96-well plate) the researchers measured an oxygen concentration of 0.55 mm above the monolayer (the minimum measurable depth of the microprobes used) of 3T3-L1 adipocytes with an OCR of 200 fmol/mm2/s, was ~ 15 mM (11 mmHg, 1.4% O2). This was significantly below the 181 mM oxygen concentration (140 mmHg, 18.4% O2) within the incubator. Consequently, the cells were being maintained in low oxygen, hypoxic conditions.
Cells respond to hypoxia primarily through the activities of oxygen-dependent prolyl-4-hydroxylase enzymes (PHD) that, in the presence of oxygen, hydroxylates proline residues on the alpha subunit of hypoxia-inducible factor (HIF-1). This targets HIF-1a for proteasomal degradation. When oxygen levels drop, the activities of PHD enzymes decrease, such that HIF-1a is no longer degraded. HIF1a then binds to HIF-1b. This heterodimer then translocates to the nucleus and initiates a transcriptional response that shifts the cell from aerobic to anaerobic metabolism.
A characteristic response to low oxygen availability is an increase in glycolysis resulting in the generation of lactate from pyruvate, and a reduction in mitochondrial oxidative phosphorylation. The culture medium from differentiated 3T3-L1 cells maintained under standard conditions was shown to contain high levels of lactate, consistent with the cells being highly glycolytic. Furthermore, such cells will exhibit a modified gene transcription profile indicative of hypoxia.
Tan et al demonstrated that lowering the volume of the medium, to change the depth by only a few mm, dramatically increased the oxygen concentration at the cell monolayer. This also dramatically reduced the levels of lactate in the medium and reduced the HIF-1-driven transcriptional response. In the 96-well plates, lowering the volume of the medium, from 100 mL to 33 mL, resulted in a doubling of the 3T3-L1 adipocyte OCR, and an increase in the oxygen concentration 0.55 mm above the monolayer to 73 mM (56 mmHg, 7.4% O2). This level of oxygen also improved adipocyte function and is comparable to the oxygen concentration measured in vivo for white adipose tissue in mice of ~ 80 mM (60 mmHg, 7.9% O2).
As noted by the authors, the study by Tan et al (2024) highlights the need to report the actual oxygen concentration that cell monolayers experience in culture, rather than the oxygen level in the incubator. Furthermore, together with the volume of the medium, the number of cells dramatically impacts the pericellular oxygen level - which in turn regulates cell function, demonstrating the need for consistency and reporting of volumes of medium and cell numbers used in experiments to ensure reproducibility.
The concentrations of oxygen in healthy human tissues range from 32 mM (23mmHg, 3% O2) to 74 mM (53 mmHg, 7% O2), whereas in tumours the oxygen levels are significantly reduced to about 3 mM (2.5mmHg, 0.3% O2) to 44 mM (32 mmHg, 4.2% O2) (McKeown 2014). Consequently, ensuring that the pericellular oxygen levels of cell lines grown in culture match the in vivo levels of the relevant tissue, or disease state, will impact experimental results, and their relevance to the tissue being studied. Furthermore, monitoring and maintaining oxygen levels and cell numbers across cell culture experiments should lead to greater reproducibility.
IMAGE The effect of increasing oxygen supply to cultured cells by lowering media depth CREDIT: Tan et al. Creative Commons
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