Mol. Hum. Reprod. Advance Access originally published online on September 28, 2006
Molecular Human Reproduction 2006 12(11):653; doi:10.1093/molehr/gal081
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Letter to the editor |
What is the appropriate oxygen tension for in vitro culture?
Division of Surgery, Oncology, Reproductive Biology and Anaesthetics, Institute of Reproductive and Developmental Biology, Imperial College London, Hammersmith Hospital, London, UK
1 To whom correspondence should be addressed at: Division of Surgery, Oncology, Reproductive Biology and Anaesthetics, Institute of Reproductive and Developmental Biology, Imperial College London, Hammersmith Hospital, Du Cane Road, London W12 0NN, UK. E-mail: mark.sullivan{at}imperial.ac.uk
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.
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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.
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References |
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 Top References |
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Ceste M (2005) Oxygen in the cultivation of stem cells. Ann N Y Acad Sci 1049,1–8.[CrossRef][Web of Science][Medline]
Guérin P, El-Mouatassim S and Ménézo Y (2001) Oxidative stress and protection against reactive oxygen species in the pre-implantation embryo and its surroundings. Hum Reprod Update 7,175–189.
Jauniaux E, Watson AL, Hempstock J, Bao YP, Skepper JN and Burton GJ (2000) Onset of maternal arterial blood flow and placental oxidative stress. A possible factor in human early pregnancy failure. Am J Pathol 157,2111–2122.
Jauniaux E, Watson A and Burton G (2001) Evaluation of respiratory gases and acid–base gradients in human fetal fluids and uteroplacental tissue between 7 and 16 weeks’ gestation. Am J Obstet Gynecol 184,998–1003.[CrossRef][Web of Science][Medline]
Karagenc L, Sertkaya Z, Ciray N, Ulug U and Bahceci M (2004) Impact of oxygen concentration on embryonic development of mouse zygotes. Reprod Biomed Online 9,409–417.[Web of Science][Medline]
Miller RK, Genbacev O, Turner MA, Aplin JD, Caniggia I and Huppertz B (2005) Human placental explants in culture: approaches and assessments. Placenta 26,439–448.[CrossRef][Web of Science][Medline]
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