What is the appropriate oxygen tension for in vitro culture?

Sir, In vitro culture is generally performed at oxygen tensions that are close to atmospheric (∼20%). Consideration of in vivo oxygen tensions suggests that 10–13% oxygen is the maximum that should be used, and for some tissues (e.g. placenta), lower levels are physiological. The detailed study of cells and tissues from humans and other organisms has been a key component of our greatly increased understanding during recent decades. Routine culture conditions have been developed, and for the majority of systems, the standard is 37°C in a defined culture medium under a humidified atmosphere of 5% CO2 : 95% air. Modern culture incubators have been designed with these criteria in mind, and they provide a reliable and robust environment for biomedical studies. Under these conditions, the partial pressure of O2 (pO2) will be ∼150 mmHg, which is equivalent to ∼20% O2 (see Table I), with the remainder of the gas in the incubator consisting primarily of nitrogen and water vapour. The pO2 of human blood normally ranges between 75 and 100 mmHg, which is equivalent to O2 gas levels of 10–13% (Table I). Routine tissue culture therefore subjects cells and tissues to pO2 levels that are substantially greater than even blood with maximum O2 saturation, and this is without considering that tissue pO2 levels will be lower than those in blood. Gaseous percentages and partial pressures of nitrogen, oxygen and water under defined conditions Gaseous percentages and partial pressures of nitrogen, oxygen and water under defined conditions The importance of such considerations is shown by work on the human placenta, demonstrating that for the first 10 weeks of pregnancy, pO2 is ∼25 mmHg, rising to about 60–65 mmHg from 12 weeks of pregnancy until term (40 weeks) (Jauniaux et al., 2000, 2001). Equivalent oxygen levels for culture are 3 and 8%, respectively, and a recent review (Miller et al., 2005) has strongly suggested that these be adopted for all placental culture. Other systems in which there is clear evidence of the deleterious effects of 20% oxygen during in vitro culture include stem cells (reviewed by Ceste, 2005) and embryos (Guérin et al., 2001; Karagenc et al., 2004). On the basis of these observational and theoretical considerations, we suggest that virtually all routine tissue cultures, and not only many investigations on the placenta, are currently being performed under hyperoxic conditions that may not reflect in vivo physiology. With the importance of oxygen in energy metabolism, redox reactions and free-radical-mediated events (both physiological and pathophysiological), consideration should be given to using O2 levels that reflect the in vivo conditions of no more than 13% and perhaps in the range from 8% (placental) to 10% (calculated, Table I) as reflecting in vivo normoxia.