Defining Physiological Normoxia for Improved Translation of Cell Physiology to Animal Models and Humans

Abstract

The extensive oxygen gradient between the air we breathe (Po2 ~21 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 O2 levels in which to culture cells, coupled with a lack of the technology to replicate and maintain a physiological O2 environment in vitro, has hindered addressing this issue thus far. In this review, we aim to address the current understanding of tissue Po2 distribution in vivo and summarize the attempts made to replicate these conditions in vitro. The state-of-the-art techniques employed to accurately determine O2 levels, as well as the issues associated with reproducing physiological O2 levels in vitro, are also critically reviewed. We aim to provide the framework for researchers to undertake cell culture under O2 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.

Extended hypoxia‐mediated H2S production provides for long‐term oxygen sensing

“…While the vast majority of in vitro metabolic experiments are performed under ambient conditions, that is, 21% O 2 , which has an approximate Po 2 of 150 mm Hg at 37°C, it is clear that these conditions are hyperoxic for most cells . Even normoxia (or more appropriately physioxia) will vary depending on the environment inhabited by cells in situ.…”

Physioxic human cell culture improves viability, metabolism, and mitochondrial morphology while reducing DNA damage

“…The benefit of culturing human cells in physioxia is not a new concept, but the molecular and cellular biology behind this phenomenon has been largely underappreciated (3). Our group has previously shown that eukaryotic initiation factor 4 E type 2–directed translation initiation, thought to only function in hypoxia, was active in the low‐to‐mid range of physioxia (17).…”

“…Indeed, we reveal a range of O 2 availability that simultaneously reduces the negative and increases the positive characteristics we tested: 5–8% O 2 in U87MG, HRPTEC, and HEK293 and a narrower range of 8% O 2 in MCF7. This range closely mirrors the mean partial pressure of oxygen range of human tissues and organs, including venous blood (5.3% O 2 ), lung (5.6% O 2 ), skin (4.6% O 2 ), brain (4.4% O 2 ), intestine (7.6% O 2 ), liver (5.4% O 2 ), kidney (9.5% O 2 ), muscle (3.8% O 2 ), and bone marrow (6.4% O 2 ) (1, 3). Of note, HRPTEC and MCF7 did not display great viability and metabolism advantages in physioxia relative to normoxia, even though normoxic DNA strand breaks and antioxidant and DNA damage gene expression were higher in both and oxidative DNAand RNA lesions were more prevalent in HRPTEC.…”

“…Cells are routinely maintained in nutrient‐rich medium in temperature‐controlled CO 2 incubators receiving filtered ambient air (21% O 2 ). Because <24 h are required for dissolved O 2 in the medium to equilibrate with ambient air (4), human cells are rapidly exposed to much higher levels of oxygen than they would be within the body [reviewed in (3)]. There is historical evidence that mammalian cells grow more rapidly when cultured at lower O 2 relative to normoxia.…”

“…Multicellular organisms balance oxygen delivery and toxicity by having oxygen pass through several barriers (e.g., lungs and vasculature) before delivery to cells and mitochondria. At each step of the oxygen cascade, the tension gradually decreases from 160 mmHg (atmospheric pressure or 21% O 2 ) to ,9 mmHg [mitochondrial O 2 tension or ,1.3% O 2 (1)(2)(3)]. This oxygen cascade preserves inside the host cell the primordial anoxic conditions of Earth for optimal function of the mitochondria, a proteobacterial remnant.…”