"Should We Call Physiologic Oxygen Hypoxia, Normoxia, Physioxia, or Something Else?"
Being able to replicate research results is dependent on having the same conditions each time. Continue reading to learn about the factors to consider when classifying room air oxygen levels.
How we describe oxygen in the microenvironment is important.
The use of physiologically relevant oxygen for in vitro cell culture is increasingly essential as cells grown in vitro become more clinically important. Oxygen levels are a critical cell parameter, just like carbon dioxide or temperature. Hyperoxia simply means too much oxygen, Normoxia means normal amounts, and Hypoxia means too little.
The term “Hypoxia” is used in two different frames of reference.
In the scientific literature, hypoxia is often used to describe physiologic oxygen levels that are lower than room air. Other researchers use same term for low oxygen conditions such as ischemia that are pathophysiologic. In these conditions, oxygen levels are too low for that particular tissue type in situ.
This has understandably led to confusion. With two different frames of reference for what is normal oxygen, the room and the body, we end with two different fundamental meanings for what is hypoxic. One recent review by Jež et al., has a great discussion of the use of the term . They chose to use the term “physiologically-relevant oxygen levels” because of this problem.
Oxygen Levels in vivo are much lower than those in the atmosphere
Tissue oxygen is lower than atmospheric air, which is about 21% at sea level. Because inspired oxygen mixes with gases in the airway immediately upon entering the body, oxygen levels in the airways and lungs are lower than the room. Once transported into the blood stream by the lungs, oxygen as a percentage falls sharply (10-13%). As oxygen is removed from the blood for use by tissues, oxygen falls more, to about 5% in venous blood. The farther the cells are away from blood vessels, the lower the oxygen level is in normal, healthy tissue.
Cell types that are clinically important to culture in vitro come from environments that are low in oxygen in vivo.
Cell types cultured for cellular therapies or regenerative medicine applications are derived from tissues that are normally low in oxygen in vivo. Bone marrow Hematopoietic Stem Cells (HSC) are found in the endosteal region next to the inside surface of the bone where blood vessels are small and the blood flow rate is slow. The only HSC that are able to produce all types of blood cells in serially transplanted recipients come from areas with extremely low oxygen levels .
Poorly vascularized tissues like cartilage are also profoundly low in oxygen as a normal and healthy condition . So it makes sense that chondrocytes grown in vitro as experimental treatments for arthritic joints function better when cultured at lower oxygen levels . See our recent post “What Oxygen Level Should I use for My Cells In Vitro?” for more resources on translating relevant in vivo oxygen levels for in vitro use.
How much does oxygen really matter in vitro?
Some clinically important cells types are extremely sensitive to oxygen levels. Hematopoietic stem cells (HSC) from cord blood or from bone marrow, for instance, change functionally at 3% oxygen. Below that level, they don’t proliferate. HSC need at least 3% to form colonies, but as oxygen levels increase above 3%, the more conditions favor differentiation rather than proliferation . As published earlier this year, broken oxygen conditions (handling cells in a room air BSC) dramatically cuts the yield of HSC from bone marrow or cord blood preparations . Mesenchymal stromal/stem cells are likewise exquisitely sensitive to oxygen levels in vitro. (See our recent post on the importance of using mesenchymal stem cells for regenerative medicine to learn more.)Oxygen levels in vitro can mean the difference between quiescence, proliferation and differentiation in many different cell types.
With two different frames of reference for discussing changes in oxygen levels, relative terms become confusing
Room air oxygen is often called Normoxic, but there is nothing normal about room air oxygen in vivo.
Normoxic is also used to describe normal tissue oxygen levels, which vary widely but are lower than room air. This makes room air Hyperoxic.
Hypoxic is also often used to describe tissue oxygen because it is lower than atmospheric oxygen levels, but this is the normal state of most in vivo tissues.
Hypoxic is also used to describe tissues with less oxygen than they have need. This is a pathologic state, not normal at all.
Hyperoxic is an oxygen level that is too high, but higher than what? Room air or normoxic tissue oxygen, which is a broad range of levels across normal tissues?
Relative terms don’t make any sense biologically when they are framed with room air oxygen levels as the reference point and they come into direct conflict when trying to compare tissue types that have different physiologic origins.
Is 5% O2 low (hypoxic), high (hyperoxic), or just right (normoxic) for my cells?
For upper airway epithelial cells, 5% oxygen is hypoxic.
For chondrocytes from cartilage, 5% oxygen is hyperoxic.
For deep tissue venous blood, 5% oxygen is normoxic, but venous blood returned to the lungs rapidly increases in oxygen to about 10%.
What impact does oxygen terminology have upon how we discuss cells?
The impact is huge when we talk about cells which need physiologic simulation in culture. In pathophysiologic conditions like ischemia, tissues can be starved for oxygen, so if you are calling your adipose-derived mesenchymal stem cells hypoxic at 3-5% oxygen, which is normal or normoxic for them , what do you call their state if you want to study them at pathologically low oxygen levels? Hypo-hypoxic? There is nowhere left to go in the relative terminology if it is framed by the room air oxygen level standard.
With the Cytocentric approach, we take the cells’ point of view and use in vivo or physiologic oxygen as the frame of reference.
One of the core Cytocentric Principles is that, Cells Need Physiologic Simulation, which means it is appropriate to use a physiologically relevant framework for describing the cellular microenvironment, even in vitro. Using room air oxygen levels as a frame of reference for cells is fundamentally a Peoplecentric point of view, thinking primarily of what people experience as environmental conditions in the room, rather than what tissues experience in situ.
One cell’s hypophysioxia is another cell’s hyperphysioxia
The use of “physiologically-relevant oxygen levels”, as a term, is a great way to think about and discuss these variable oxygen states. The shorter terms Physioxia and Physioxic  are also an improvement over Hypoxia, in that they place a clear emphasis on the use of an appropriate physiologic frame of reference for in vivo oxygen levels. In the context of the biology of HIF proteins, biologic oxygen sensors which respond at the molecular level to oxygen changes, it can make sense to use relative terms like Hyperphysioxic or Hypophysioxic. These terms bring the referenced oxygen state to the cells’ level rather than the room’s level.
However, for the greatest clarity when discussing the oxygen microenvironment for critically important cell cultures, state the actual numeric oxygen levels as often as possible. As far as the state of a tissue being described, any one value for oxygen partial pressure may mean something very different to other researchers, but actual numerical values are clear.
We recommend, for clarity, using specific oxygen levels when reporting your results and avoiding relative terms like “hypoxia” that may or may not use non-physiologic room air as a frame of reference.
1. 1. Jež, M., et al., Concise Review: The Role of Oxygen in Hematopoietic Stem Cell Physiology. Journal of cellular physiology, 2015. 230(9): p. 1999-2005.
2. 2. Levesque, J.P. and I.G. Winkler, Hierarchy of immature hematopoietic cells related to blood flow and niche. Curr Opin Hematol, 2011. 18(4): p. 220-5.
3. 3. Gibson, J.S., et al., Oxygen and reactive oxygen species in articular cartilage: modulators of ionic homeostasis.Pflugers Arch, 2008. 455(4): p. 563-73.
4. 4. Buckley, C.T., T. Vinardell, and D.J. Kelly, Oxygen tension differentially regulates the functional properties of cartilaginous tissues engineered from infrapatellar fat pad derived MSCs and articular chondrocytes.Osteoarthritis Cartilage, 2010. 18(10): p. 1345-54.
5. 5. Ivanovic, Z., et al., Simultaneous Maintenance of Human Cord Blood SCID‐Repopulating Cells and Expansion of Committed Progenitors at Low O2 Concentration (3%). Stem Cells, 2004. 22(5): p. 716-724.
6. 6. Mantel, C.R., et al., Enhancing Hematopoietic Stem Cell Transplantation Efficacy by Mitigating Oxygen Shock.Cell, 2015. 161(7): p. 1553-65.
7. 7. Carreau, A., et al., Why is the partial oxygen pressure of human tissues a crucial parameter? Small molecules and hypoxia. Journal of cellular and molecular medicine, 2011. 15(6): p. 1239-1253.
About the Author
Alicia D Henn, PhD, MBA
Alicia Henn has been the Chief Scientific Officer of BioSpherix, Ltd for two years. Previously, she was a researcher at the Center for Biodefense Immune Modeling in Rochester, NY. Alicia obtained her PhD in molecular pharmacology and cancer therapeutics from Roswell Park Cancer Institute in Buffalo, NY and her MBA from the Simon School at University of Rochester in Rochester, NY.