cytocentric visionaries ju p1Cytocentric Visionaries: Dr. Jim Uniacke, University of Guelph

Cells are Vastly Different Under Physiologic Oxygen than in Room Air

James Uniacke, PhD, is an Assistant Professor in the Department of Cellular and Molecular Biology at the University of Guelph where his lab studies protein translation machinery.

Here, Alicia talks with Jim about his group’s recent publication in JBC, “Human Cells Cultured under Physiological Oxygen Utilize Two Cap-binding Proteins to Recruit Distinct mRNAs for Translation.” [1]  This interview discussing how differing molecular mechanisms can result from varied physiological conditions was edited for length.

AH: This paper was remarkable for its unusually clear language, including the use of the term “physioxia.” The meaning of the term “hypoxia” is often blurred between pathophysiologically low oxygen and normal physiologic low oxygen. You not only drew attention to the correct use of the terms hypoxia and physioxia, but described critical differences in the protein translation mechanisms active in cells under these two fundamentally different states.

JU: In the scientific community, hypoxia is about 1% to 2.5% oxygen, but we saw some recent papers reporting the normal oxygen levels in our organs, and they were surprisingly close to hypoxia. The mean oxygenation of our brain, for example, is roughly 4 to 5%. Physiologic oxygen, called physioxia, is much closer to hypoxia than “normoxia”, the room air (closer to 20% oxygen) that most labs culture their cells in.

So we examined physioxic cells for a new protein synthesis mechanism that we had previously discovered in hypoxic cells. We discovered that, indeed, the method of translation initiation that we study, an alternative to what is in our textbooks, is functional in physioxia.

It really surprised us that there are fundamental biological processes, like protein synthesis, that could be functioning differently than we understood from room air based research. Some pathways that are not known to be active in normal physiologic situations are actually normal. Not only is this new method of translation initiation normal in physiological conditions, but it suggests that many other fundamental biological pathways might be different under physioxia.

This is a very important area of research, considering oxygen as a parameter in cell culture to truly represent physiological conditions.

AH: Why do you think researchers have let oxygen fall by the wayside as a critical parameter?

JU: I did a little digging there. The dawn of human cell culture basically started when HeLa cells were cultured in the 1950s or 60s. Back then, cell culture parameters were just starting to be set, so oxygen was considered. However, HeLa cells grew fine under normoxia, or ambient air. It was more convenient to grow them in ambient air. It became a norm.

My paper showed that there are molecular pathways that actually sense oxygen and are different under low oxygen. So cells might be growing okay, but maybe they’re more stressed and maybe they’re growing more slowly.

It’s coming into fashion now because we have a greater understanding of oxygen sensing at the cellular level and at the molecular level. We’re starting to realize that if cells within our bodies are under physiologic oxygen, then they’re functioning differently. Some pathways are turned on, some pathways are turned off and the genes that are being expressed are completely different. So I think you’re going to see a greater spotlight shining on oxygen as a cell culture parameter.

AH: Agreed. The cell lines you used had been in physioxic conditions for 24 hours before use. Do you think these findings extend to in vivo where physioxia is constant?

JU: That’s a great point. Do they fully recapitulate the in vivo situation? Perhaps not. These cells are routinely cultured in normoxia until we decide to incubate them in physioxia for 24 hours. They are likely still in the process of adapting. I think this is just a start. The methods in our paper could definitely be built upon. Maybe cell lines from now on can be established in physioxia and never leave physioxia. That would be ideal.

This was just to highlight that there are some fundamental molecular pathways that are likely quite different in physioxia that we’re missing.

AH: How aware are other researchers about terms like physioxia and the functional differences of cells in that state in vitro?

JU: The scientific community is aware that physiological oxygen is much lower than what they’re culturing their cells in, but I think that they don’t think it matters.

When my paper came out and the university did a press release, it got some attention. People contacted me and said “It’s been known for awhile that physiological oxygen is lower than ambient air.” I said that’s not the point of the paper. The point of our paper is to show that there are some molecular mechanisms that are different. That should-- I don’t know if “scare” people is the word-- but cells are expressing different genes. Cells are different.

That’s what I think the scientific community doesn’t appreciate is that cells are likely vastly different when cultured under physiological conditions. If you’re studying some molecular mechanism in cell biology and you’re culturing your cells near 20% oxygen, it could be very well different than a cell that’s residing in a human body.

AH: Do you see application of your findings in enhancing scientific reproducibility?

JU: I agree with that. I think when we’re starting to translate our findings to what’s happening in people, we’ve got to think of physioxia. Cell culture is the starting point for a lot of disease research and testing drugs, and that could be completely different under physioxia. I know under hypoxia, different importers and exporters and transporters are all differently expressed. So culturing your cells under normoxia and testing if a drug works on a certain pathway for example, I don’t see how that can translate to in vivo. We need to get the word out.

AH: So what do you think the next step is for your research moving on from here?

JU: Instead of focusing on molecular pathways, since oxygen is associated with reactive oxygen species, we are taking a step back and looking at stress. If we can show that cells under normoxia are expressing stress response genes and are being damaged by reactive oxygen species, it might be what the scientific community wants to see.

AH: Anything else you would like to tell our readers?

JU: Don’t forget oxygen as a parameter when you’re culturing your cells. We’re fully aware that there’s CO2 in our CO2 incubators, but cells have evolved, especially eukaryotic cells, to sense and use oxygen. They’re tuned to the oxygen availability. Gene expression, in turn, is altered depending on oxygen. So whatever your research is, oxygen is going to matter.

1. Timpano, S. and J. Uniacke, Human Cells Cultured Under Physiological Oxygen Utilize Two Cap-binding Proteins to Recruit Distinct mRNAs for Tranlation. J Biol Chem, 2016.


alicia author iconAbout 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.