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
Bill S-201, the Genetic Non-Discrimination Act in Canada
Since the dawn of modern man, we as a species have been obsessed with the categorization of ourselves. The first primary way, by the obvious: skin colour, eyes, hair, genitalia. Those items easy to pick out with a glance. As the world shrank with widespread travel and discovery, we then began to subcategorize with language and religion. Of course all of these categories intermingle with each other. Unfortunately, as we discovered the differences we immediately started ranking and stereotyping groups. Being a victim of this ‘stereotyping’ in my early years, I can say it certainly made its impact on me. Flash forward to today, we still have not evolved past racial discrimination across the world but I can say without a doubt that Canada, based on my own perspective, has improved dramatically. Progress, or is it? Canada in particular has allowed a new type of discrimination to infiltrate amongst us. Other nations in Europe have recognized it and the US has even commenced dealing with it. But Canada seems to be a little further behind. Bill S-2011 is the new issue of genetic discrimination.
Genetics has really been a science for many years with its most notable point of origin Gregor Mendel (1822-1884)2, of which the term Mendelian genetics is drawn from. He bred a couple of varieties of pea plants, green and yellow peas, also tracking a number of other physical traits. Through all his work he discovered dominant and recessive genes and with this information human kind became quite proficient at being able to calculate the probability of different phenotypic (visible expression of the genes) outcomes. We will return to the use of the term “probability” in a second. As science progressed, we started to unlock the structure of DNA (the basic coding of life) and further endeavour to fully understand how this code is read and translated into proteins, tissues and systems. Canada hosts a number of prominent researchers in this arena, many concentrated with grand facilities in the Toronto Discovery District as well as all across Ontario. Since Venter et al.3 with Celera Corp. attempted a “shot-gun” mass sequencing of the human genome years ago, we have discovered a lot of changes happen after the first stage of the DNA being read. Having worked at MDS Proteomics myself, the goal with this organization was originally to map the human proteome in similar fashion to Venter/Celera but it became abundantly clear this was a much more ambitious project than the genome, and to this day, we are still a long way from completing the task. Why is this?
Circling back to the term “probability” in genetics now, it’s an important thing to understand. We use this term because we often find exceptions to a calculated model. For instance, some critically acclaimed work on a world scale performed in the arena of cystic fibrosis was conducted at the Hospital for Sick Children in Toronto by Dr Lap-Chee Tsui.4 In short, there was a known genetic sequence (gene) on chromosome 7 (of 23) known to have some strong hand in predicting whether a person has CF or not, but with this given sequence there was a full range of disease severity. Dr Tsui’s (and many other’s) further work went on to discover there are many complexities that changed how this gene was expressed and the variety of impacts this differential expression had on net result (and this work continues to this day revealing more and more). To summarize, just because you have the particular marker characterizing a particular gene, it doesn’t mean you express it the same way. The human body, and any living model for that matter, is complex and interactions with other genes or systems cannot always be predicted. One mutation putting you at a disadvantage could give you an advantage in another scenario. An example of this is sickle cell anemia sufferers of certain ages are generally immune to the effects of Malaria due to the fact that their mutated red blood cells prevent the malaria parasite from effectively propagating.5
Armed with all these examples and some knowledge above, lets look at insurance companies doing business in Canada. When you make arrangements to take on new life insurance policy or critical illness plan they go through an extensive questionnaire, they can draw blood, cheek cells by swab, possibly urine, etc. What they do next with these samples is send them to a regional lab and have your samples tested against a number of markers well as the standard drug screens and metabolites. That’s why Bill S-201 is so important. It’s one thing to make a claim of being smoke or drug free and then get tested to reveal the contrary — you have a choice in this case and you made the wrong one. But, if these samples are being tested for particular markers (some may not even be outlined) and evaluations are being made on your genetic make up, is this fair? You could be an exemplary athlete with markers for heart disease but because you are constantly monitored, exercise and eat right, is it fair that you are judged based on what that marker typically results in with your average human being?
We are a long way from being able to nail down final outcome based on genetic predisposition. Using this type of information to guide or make one aware of possible scenarios is the best use for this type of analysis and the knowledge that comes with it is great when determining wide scale calls to action in health care. But never should it be used to discriminate. Bill S-201 has not passed as of yet but has received much press and shows promise to be passed. Let’s hope Canada will also take a stand and limit this discrimination which is already occurring.
This post was written by Ranjan Mukherjee
1. Bill S-201 :This enactment prohibits any person from requiring an individual to undergo a genetic test or disclose the results of a genetic test as a condition of providing goods or services to, entering into or continuing a contract with, or offering specific conditions in a contract with the individual. Exceptions are provided for medical practitioners and researchers, as well as for insurance providers in respect of high-value insurance contracts if provincial laws expressly permit a requirement that existing genetic test results be disclosed.The enactment amends the Canada Labour Code to protect employees from being required to undergo or to disclose the results of a genetic test, and provides employees with other protections related to genetic testing and test results. It also amends the Canadian Human Rights Act to prohibit discrimination on the ground of genetic characteristics. http://openparliament.ca/bills/41-2/S-201/↩
2. Mendel, Gregor. 1866. Versuche über Plflanzenhybriden. Verhandlungen des naturforschenden Vereines in Brünn, Bd. IV für das Jahr 1865, Abhandlungen, 3–47.↩
3. The Sequence of the Human Genome J. Craig Venter,1 * Mark D. Adams,1 Eugene W. Myers,1 Peter W. Li,1 Richard J. Mural,1 Granger G. Sutton,1 Hamilton O. Smith,1 Mark Yandell,1 Cheryl A. Evans,1 Robert A. Holt,1 Jeannine D. Gocayne,1 Peter Amanatides,1 Richard M. Ballew,1 Daniel H. Huson,1 Jennifer Russo Wortman,1 Qing Zhang,1 Chinnappa D. Kodira,1 Xiangqun H. Zheng,1 Lin Chen,1 Marian Skupski,1 Gangadharan Subramanian,1 Paul D. Thomas,1 Jinghui Zhang,1 George L. Gabor Miklos,2 Catherine Nelson,3 Samuel Broder,1 Andrew G. Clark,4 Joe Nadeau,5 Victor A. McKusick,6 Norton Zinder,7 Arnold J. Levine,7 Richard J. Roberts,8 Mel Simon,9 Carolyn Slayman,10 Michael Hunkapiller,11 Randall Bolanos,1 Arthur Delcher,1 Ian Dew,1 Daniel Fasulo,1 Michael Flanigan,1 Liliana Florea,1 Aaron Halpern,1 Sridhar Hannenhalli,1 Saul Kravitz,1 Samuel Levy,1 Clark Mobarry,1 Knut Reinert,1 Karin Remington,1 Jane Abu-Threideh,1 Ellen Beasley,1 Kendra Biddick,1 Vivien Bonazzi,1 Rhonda Brandon,1 Michele Cargill,1 Ishwar Chandramouliswaran,1 Rosane Charlab,1 Kabir Chaturvedi,1 Zuoming Deng,1 Valentina Di Francesco,1 Patrick Dunn,1 Karen Eilbeck,1 Carlos Evangelista,1 Andrei E. Gabrielian,1 Weiniu Gan,1 Wangmao Ge,1 Fangcheng Gong,1 Zhiping Gu,1 Ping Guan,1 Thomas J. Heiman,1 Maureen E. Higgins,1 Rui-Ru Ji,1 Zhaoxi Ke,1 Karen A. Ketchum,1 Zhongwu Lai,1 Yiding Lei,1 Zhenya Li,1 Jiayin Li,1 Yong Liang,1 Xiaoying Lin,1 Fu Lu,1 Gennady V. Merkulov,1 Natalia Milshina,1 Helen M. Moore,1 Ashwinikumar K Naik,1 Vaibhav A. Narayan,1 Beena Neelam,1 Deborah Nusskern,1 Douglas B. Rusch,1 Steven Salzberg,12 Wei Shao,1 Bixiong Shue,1 Jingtao Sun,1 Zhen Yuan Wang,1 Aihui Wang,1 Xin Wang,1 Jian Wang,1 Ming-Hui Wei,1 Ron Wides,13 Chunlin Xiao,1 Chunhua Yan,1 Alison Yao,1 Jane Ye,1 Ming Zhan,1 Weiqing Zhang,1 Hongyu Zhang,1 Qi Zhao,1 Liansheng Zheng,1 Fei Zhong,1 Wenyan Zhong,1 Shiaoping C. Zhu,1 Shaying Zhao,12 Dennis Gilbert,1 Suzanna Baumhueter,1 Gene Spier,1 Christine Carter,1 Anibal Cravchik,1 Trevor Woodage,1 Feroze Ali,1 Huijin An,1 Aderonke Awe,1 Danita Baldwin,1 Holly Baden,1 Mary Barnstead,1 Ian Barrow,1 Karen Beeson,1 Dana Busam,1 Amy Carver,1 Angela Center,1 Ming Lai Cheng,1 Liz Curry,1 Steve Danaher,1 Lionel Davenport,1 Raymond Desilets,1 Susanne Dietz,1 Kristina Dodson,1 Lisa Doup,1 Steven Ferriera,1 Neha Garg,1 Andres Gluecksmann,1 Brit Hart,1 Jason Haynes,1 Charles Haynes,1 Cheryl Heiner,1 Suzanne Hladun,1 Damon Hostin,1 Jarrett Houck,1 Timothy Howland,1 Chinyere Ibegwam,1 Jeffery Johnson,1 Francis Kalush,1 Lesley Kline,1 Shashi Koduru,1 Amy Love,1 Felecia Mann,1 David May,1 Steven McCawley,1 Tina McIntosh,1 Ivy McMullen,1 Mee Moy,1 Linda Moy,1 Brian Murphy,1 Keith Nelson,1 Cynthia Pfannkoch,1 Eric Pratts,1 Vinita Puri,1 Hina Qureshi,1 Matthew Reardon,1 Robert Rodriguez,1 Yu-Hui Rogers,1 Deanna Romblad,1 Bob Ruhfel,1 Richard Scott,1 Cynthia Sitter,1 Michelle Smallwood,1 Erin Stewart,1 Renee Strong,1 Ellen Suh,1 Reginald Thomas,1 Ni Ni Tint,1 Sukyee Tse,1 Claire Vech,1 Gary Wang,1 Jeremy Wetter,1 Sherita Williams,1 Monica Williams,1 Sandra Windsor,1 Emily Winn-Deen,1 Keriellen Wolfe,1 Jayshree Zaveri,1 Karena Zaveri,1 Josep F. Abril,14 Roderic Guigo´,14 Michael J. Campbell,1 Kimmen V. Sjolander,1 Brian Karlak,1 Anish Kejariwal,1 Huaiyu Mi,1 Betty Lazareva,1 Thomas Hatton,1 Apurva Narechania,1 Karen Diemer,1 Anushya Muruganujan,1 Nan Guo,1 Shinji Sato,1 Vineet Bafna,1 Sorin Istrail,1 Ross Lippert,1 Russell Schwartz,1 Brian Walenz,1 Shibu Yooseph,1 David Allen,1 Anand Basu,1 James Baxendale,1 Louis Blick,1 Marcelo Caminha,1 John Carnes-Stine,1 Parris Caulk,1 Yen-Hui Chiang,1 My Coyne,1 Carl Dahlke,1 Anne Deslattes Mays,1 Maria Dombroski,1 Michael Donnelly,1 Dale Ely,1 Shiva Esparham,1 Carl Fosler,1 Harold Gire,1 Stephen Glanowski,1 Kenneth Glasser,1 Anna Glodek,1 Mark Gorokhov,1 Ken Graham,1 Barry Gropman,1 Michael Harris,1 Jeremy Heil,1 Scott Henderson,1 Jeffrey Hoover,1 Donald Jennings,1 Catherine Jordan,1 James Jordan,1 John Kasha,1 Leonid Kagan,1 Cheryl Kraft,1 Alexander Levitsky,1 Mark Lewis,1 Xiangjun Liu,1 John Lopez,1 Daniel Ma,1 William Majoros,1 Joe McDaniel,1 Sean Murphy,1 Matthew Newman,1 Trung Nguyen,1 Ngoc Nguyen,1 Marc Nodell,1 Sue Pan,1 Jim Peck,1 Marshall Peterson,1 William Rowe,1 Robert Sanders,1 John Scott,1 Michael Simpson,1 Thomas Smith,1 Arlan Sprague,1 Timothy Stockwell,1 Russell Turner,1 Eli Venter,1 Mei Wang,1 Meiyuan Wen,1 David Wu,1 Mitchell Wu,1 Ashley Xia,1 Ali Zandieh,1 Xiaohong Zhu1 T H E H UMAN G ENOME 1304 16 FEBRUARY 2001 VOL 291 SCIENCE↩
4. 1985 Tsui L, Buchwald M, Barker D, Braman JC, Knowlton R, Schumm JW, Eiberg H, Mohr J, Kennedy D, Plavsic N, et al. Cystic fibrosis locus defined by a genetically linked polymorphic DNA marker. Science 1985; 230:1054-1057.↩
5. Protective Effects of the Sickle Cell Gene Against Malaria Morbidity and Mortality. Aidoo M, Terlouw DJ, Kolczak MS, McElroy PD, ter Kuile FO, Kariuki S, Nahlen BL, Lal AA, Udhayakumar V. Lancet 2002; 359:1311-1312.↩
Why buy from the little guy?
This question is often asked by representation from larger companies: “why take a risk buying from a small company”? They imply that these small lab suppliers will disappear in the night, provide mediocre service or have less capacity to supply the consumer’s total needs. I have a number of statements contrary to this philosophy, but I will use a recent example of a client’s relayed experience.
The client had invested in some centrifuges from a manufacturer who is very reputable in the industry and the units were actually bought from a very large distributor. After 3 consistent failures experienced by the client, they demanded the distributor and manufacturer explain themselves and offer a solution that worked. After much lip service, they decided to offer him 20% off the next unit he purchased! This was at a very prestigious University in Canada. Can you guess where the client insisted that they shove the offer? But in all that he lost, he paid top dollar for bottom dollar product and service.
The point of all this is that we, the customer, are not important enough for the bigger companies to allocate the resources to help us. The big corporations are a slave to the ‘shareholder’ and in that, spend more time in activities of trimming staff, hiring more managers, cutting costs (making things cheaper) and scrutinizing metrics to deliver dollars on their timelines – not the client’s . This then looks like “strategic” moves to the shareholders and keeps or encourages investment. This typical process strays further from the client or customer as they are merely considered one transaction and less worthy of tracking.
The little guy operates on a different track. Too small for major investment, the primary source of revenue is the client! With little budget to advertise like the big corporations, the small lab suppliers rely on positive transactions, word of mouth and decent prices to carry on. Often, the little guy also locates and secures novel products overlooked by big business being deemed ‘too little’ for potential revenue. And to address the ‘disappear into the night’ comment, sure, this is possible with small companies. But how many larger ones have filed chapter 11 or been bought out by larger companies who either bury their acquired products or make no claim to responsibility of the existing install base post acquisition. Bottom line: bigger is not better.
By the time I had happened upon the door of the aforementioned client, he was quite upset and still needed a centrifuge. Although I was working with a small manufacturer who makes a limited amount of good quality products, the brand name is unknown and the client expressed his concern over having a similar experience as above. My response was simple. Though no one can foresee the future, the unit is almost half price, carries the same warranty and the organization and manufacturer are both reliant on a positive experience to help move more product. How much worse could he end up than he already was?
Next time you are in this conundrum, consider how many of your dollars go towards the actual product versus the endless layers to over-promote, warehouse and ship the product. Compound that with having many people make mistakes, sending your stuff awry or simply shipping the wrong item and trying to correct all that by phone! This is where the little guy is worth it!
This post was written by Ranjan Mukherjee