The extensive oxygen gradient between the air we breathe (PO 2~2 1 kPa) and its ultimate distribution within mitochondria (as low as~0.5-1 kPa) is testament to the efforts expended in limiting its inherent toxicity. It has long been recognized that cell culture undertaken under room air conditions falls short of replicating this protection in vitro. Despite this, difficulty in accurately determining the appropriate O 2 levels in which to culture cells, coupled with a lack of the technology to

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... plicate and maintain a physiological O 2 environment in vitro, has hindered addressing this issue thus far. In this review, we aim to address the current understanding of tissue PO 2 distribution in vivo and summarize the attempts made to replicate these conditions in vitro. The state-of-the-art techniques employed to accurately determine O 2 levels, as well as the issues associated with reproducing physiological O 2 levels in vitro, are also critically reviewed. We aim to provide the framework for researchers to undertake cell culture under O 2 levels relevant to specific tissues and organs. We envisage that this review will facilitate a paradigm shift, enabling translation of findings under physiological conditions in vitro to disease pathology and the design of novel therapeutics.