Michael T Kane, NUI Galway, Ireland

https://doi.org/10.36866/pn.118.7

Blood equilibrated with air under normal laboratory conditions contains about 20% O2 (v/v). As every first-year student in physiology learns, given the very low solubility of oxygen in aqueous solutions, this concentration of oxygen in normal arterial blood is only possible due to the oxygen-carrying power of haemoglobin.

Thus reports in some publications (see below) of levels of up to 20% oxygen in haemoglobin-free biological fluids such as cell culture media are both meaningless, in the absence of units, and grossly misleading. The true figure is of the order of 0.4% O2 (v/v) based on the fact that solubility of oxygen in plasma is given as 2.09 ml per 100 ml plasma at 38°C and 1 atm (760 mmHg) pressure of oxygen in the gas phase.¹ Oxygen is both essential to cells for their viability and energy production but is at the same time dangerous to those same cells because high concentrations can lead to the production of reactive oxygen species and free radicals, which damage cells. This is referred to as the Oxygen Paradox of Davies.² Therefore, it is of vital importance that the terminology used to report the oxygen concentration in cell culture be always unambiguous and correct. This letter provides examples of errors in this context and explains how such egregious errors may have arisen.

In a 2012 paper on chondrogenic cell culture in In Vitro Cellular & Developmental Biology– Animal, one oxygen concentration in the culture medium is given as 18.99%. In another paper on human trophoblast culture in Placenta in 2013, it is stated that the concentration of oxygen resembled that in the ambient atmosphere. Another paper on HeLa cell culture in Cell Biology International in 2016 reports dissolved oxygen concentrations up to 18%. This kind of error is not confined to culture studies. A paper on the oxygen concentration of human ovarian follicular fluid in Human Reproduction in 1997 reports “average percentage dissolved oxygen” figures of 1.7 to 4.2. A paper on cattle follicular fluids in Theriogenology in 2008 reports follicular fluid oxygen concentrations of 6.9 % and 7.3%.

Even in an otherwise excellent and very useful paper entitled “Dissolved oxygen concentration in culture medium: assumptions and pitfalls”, there is the comment “in this study, dissolved oxygen levels in culture medium maintained in standard culture conditions (18% O2) measured 18%”.³

How has this erroneous terminology crept into current mainstream literature?

A minor reason is probably the general custom of referring to gas phase concentrations in gas mixtures without units e.g. 5% CO2 and not 5% CO2 (v/v). A major reason is the failure to realise that oxygen is very poorly soluble in aqueous media. However, another major reason is that much of the instrumentation generally used for measurement of dissolved oxygen in culture media or biological fluids was not originally designed for this purpose. One such device is the Jenway DO2 oxygen meter.³ Email correspondence with the suppliers of these meters informed me that “the Jenway DO2 meters were originally developed for determining the quality of environmental water systems… The meters are able to display either % oxygen saturation or % air saturation and are calibrated according to the parameter required.”

It is clear from information on the websites of other manufacturers and some email correspondence with them that most, if not all, the oxygen sensor probes available are based on a polarographic electrode or alternatively a fibre-optic device that measures PO2 which can then be translated into other units including % oxygen saturation or % air saturation but NOT usually % oxygen v/v. Thus, when papers like those cited above say that the medium has an oxygen concentration of 5%, this does not mean that the medium contains 5% v/v dissolved oxygen. Rather, it means that the medium contains the same concentration of oxygen as it would if it were fully equilibrated with a gas phase of 5% v/v oxygen, so that the PO2 of the gas phase and the fluid are exactly the same i.e. the % oxygen saturation is 5%. Based on the oxygen solubility in plasma¹ the true value of the % oxygen concentration (v/v) for 5% oxygen saturation is about 0.10% i.e. about 1/50 of the oxygen saturation value. Note that in saying v/v or vol % it is obviously understood that one is using the same volume unit for both numerator and denominator.

A crucial point or much ado about nothing?

It may seem that this question of terminology is like Shakespeare’s play Much Ado About Nothing. However, this is not correct. Firstly, in specifying culture medium levels for oxygen, which is essential for cell function but potentially toxic, the terminology used should be, as stated above, unambiguous and correct. At a time when the most recent Nobel Prize in Physiology or Medicine has been awarded to Kaelin, Ratcliffe and Semenza for their discovery of how cells sense and adapt to oxygen availability, it would seem more essential than ever that authors and journal editors report oxygen concentrations in biological fluids appropriately.

Secondly, the misuse of the terminology is not producing minor errors, it is suggesting values that are out by a factor of about 50 and clearly impossible with normal incubator gas pressures and gas phase oxygen concentrations.

Thirdly, reporting values as % oxygen saturation as used in reference 4 would be correct.⁴ However, this is a clumsy terminology for cell culture and since gas diffusion always takes place along a partial pressure or tension basis, it is preferable that measurements should be made and reported in partial pressure units such as mm Hg. Some of the oxygen sensors, but not all, can be set up to report readings in partial pressure units (mm Hg, kPa).

References

1. Pittman RN (2011). Regulation of Tissue Oxygenation. Chapter 3, The Respiratory System and Oxygen Transport. Morgan & Claypool Life Sciences San Rafael (CA). Available from: ncbi.nlm.nih.gov/books/NBK54114/
2. Davies KJA (2000). Oxidative stress, antioxidant defenses, and damage removal, repair, and replacement systems. IUBMB Life 50(4-5), 79 – 289. DOI: 10.1080/713803728
3. Newby D et al. (2005). Dissolved oxygen concentration in culture medium: assumptions and pitfalls. Placenta 26(4), 353 – 357. DOI: 10.1016/j.placenta.2004.07.002
4. Naciri M et al. (2008). Monitoring pH and dissolved oxygen in mammalian cell culture using optical sensors. Cytotechnology 57(3), 245 – 250. DOI: 10.1007/s10616-008-9160-1