Finding the new Hype in Hypoxia
Can controlled oxygen hold the key to better understanding diseases like cancer, chronic inflammation as well as increase success with processes such as IVF and stem cell therapy?
These conditions and disease may sound completely unrelated, but there is a common tie to their research: cell culture. The evolution of growing human cell lines independent of the body has been effectively done for many years now, originally starting with the HeLa cell derived from a non-consenting cancer patient, Henrietta Lacks, many years ago and the comprehension of cell biology evolved from that point until now. We have successfully cultured different cell lines to perform any number of experiments: from seeing how cells and tissues communicate, adding genetic code to produce desired proteins and even altering their behaviour as is done with stem cell work to produce different types of tissues. Applications cover a wide spectrum but predominant focus has been on the study of cancer, diabetes, heart disease, stem cell therapy and inflammatory diseases.
Culturing cells has been conducted the same way for a number of years using humidified chambers set at 37 C with piped in CO2 at about 5%. CO2 incubators rose in popularity over the years as they helped stabilize the media feeding cell lines by buffering action of CO2 being converted into HCO3-, 1 effectively prolonging a cell line. Much work has gone into finding ideal media to feed the cells and keep them relatively healthy as well. With all the concerns addressed, interestingly enough, very few considered the amount of oxygen present in and around the culture and what the effect might be. To put things in perspective, our atmosphere, at sea level contains approximately 21% Oxygen.2
Something to Consider
Now as a fellow air breathing mammal we use our lungs to capture the oxygen we need to survive and nourish our cells to perform respiration, and secondarily all the daily functions they are responsible for on a tissue and cellular level. Now consider, our most oxygen rich point in our body is our upper air way at 19.7 %3 oxygen, then to the alveoli where most of the gas exchange occurs now 14.5% 3. By the time you have approached organ tissues and bone, we are in single digits and then within individual cells just above 1%!4 The oxygenation at these points are referred to as ‘physiological normoxic’ or ‘physoxic’ which is dependent on the particular tissue you are working with but in all cases much less than 21%. Each cell in each respective tissue has evolved to work optimally with the given access to oxygen its place in the body.
By some miracle we have been able to coax various cell lines to grow, derived from various tissues of the body. We simply incubate at physiological body temp (37C), keep it moist (about 80 % RH) and pump about 5% CO2 in and hope for the best. Some are a little more enterprising and add N2 to help offset some oxygen to evoke specific responses or illicit certain behaviours from the cell, but the fair majority just monitor their CO2. Using an obvious analogy, if you pull a fish out of the water and put it on land, it will die in short order as it is not accustomed to have access to that much oxygen, nor are its systems capable of capturing it properly. Are cell cultures not the same? If you are working with skeletal muscle tissue which rarely sees more than 3% oxygen,4 how do you suppose it will react at closer to 20%? This is a condition called ‘hyperoxia’ where in a living creature will cause harm and/or death eventually. 4 Mammalian cells have some methods to cope with variation, but as a result are not operating normally when these systems are put into play.
The Dawn of the New Concept
Some early work was done by Rueckert RR and Mueller in 19605 simply discovering that nitrogen exposed (O2 eliminated) cells in culture indeed grew almost as well as those in oxygen and further, those in excess oxygen either stopped growth or declined. This incited a few papers within that same decade followed by a small number looking into this topic through the 70’s and 80’s. With the plethora of new technology being added to science, the idea took a bit of a back burner till the late 90’s when a few scientists decided O2 control was important and so was born the first hypoxia workstation produced by Ruskinn in the UK.
From this point onward a small movement has been afoot to rethink and re-examine results accepted long ago using the traditional cell sustaining methods, but, could some previously performed experiments yield new results, while carefully mimicking the environment these cells were originally drawn from?
There are a number of studies already showing that tumour growth accelerates in hypoxic conditions.7 A protein complex referred to as the HIF (hypoxia inducible factor) complex has been shown to be regulated at a transcription level 6 and is involved in triggering a number of other reactions and systems. Though we are not fully versed on the total effect, one thing is for certain, cells (and bacteria for that matter) do not behave the same when exposed to varying levels of oxygen outside of their norm. The HIF complex has further been implicated with cellular senescence and premature aging within mammalian cells, laying a large mine to start mining from! 8
Will this be the bridge to better understanding how cells work? It’s certainly worth looking into! Imagine that many experiments have had to move to animal models to replicate in-vivo conditions, but controlling oxygen may allow researchers to go further with cell cultures. It’s hard to tell the potential of considering oxygen level in research or what will and won’t be other pivotal epiphanies to the industry. With the relative ease of access to this type of oxygen controlled technology, there is likely to be a number of new discoveries hiding in the research of the past. Will hypoxia blaze a new trail to discovery? Only research will tell.
This post was written by Ranjan Mukherjee.
- Freshney, R. Ian. Culture of Animal Cells: A Manual of Basic Technique. 3rd ed. New York: Wiley-Liss, 1994: 80-84.
- Oxygen Source for figures: Carbon dioxide, NOAA Earth System Research Laboratory, (updated 2013-03). Methane, IPCC TAR table 6.1, (updated to 1998). The NASA total was 17 ppmv over 100%, and CO2 was increased here by 15 ppmv.
- Aude Carreau a, Bouchra El Hafny-Rahbi a, Agata Matejuk b, Catherine Grillon a, *, Claudine Kieda “Why is the partial oxygen pressure of human tissues a crucial parameter? Small molecules and hypoxia” J. Cell. Mol. Med. Vol 15, No 6, 2011 pp. 1239-1253
- Mach, William J.; Thimmesch, Amanda R.; Pierce, J. Thomas; Pierce, Janet D. “Consequences of Hyperoxia and the Toxicity of Oxygen in the Lung”. Nursing Research and Practice 2011: 1–7. doi:10.1155/2011/260482.)
- Rueckert RR and Mueller GC “Effect of Oxygen Tension on HeLa Cell Growth*”, Cancer Res July 1960 20; 944
- Ratcliffe P (2002). “From erythropoietin to oxygen: hypoxia-inducible factor hydroxylases and the hypoxia signal pathway”. Blood Purif. 20 (5): 445–50. doi:10.1159/000065201. PMID 12207089.
- Kumar P1, Bacchu V2, Wiebe LI2. “The Chemistry and Radiochemistry of Hypoxia-Specific, Radiohalogenated Nitroaromatic Imaging Probes. “Semin Nucl Med. 2015 Mar;45(2):122-135. doi: 10.1053/j.semnuclmed.2014.10.005.
- Amit Maity and Constantinos Koumenis “HIF and MIF—a nifty way to delay senescence?”, Published by Cold Spring Harbor Laboratory Press, Genes Dev. 2006 20: 3337-3341