I was wondering if anybody has experience conducting chronic hypoxia experiments using a hypoxic cell incubator (1% O2, 5%CO2, 37deg), and taking samples every X amount of hours?
I am struggling to demonstrate reductions in cell viability owing to hypoxia using a standard MTT assay in both primary and immortalised cardiac cell lines.
Is it possible that opening and closing the door to remove samples for each time point is conditioning the cells? O2 levels spike to around 8% after opening the door, but quickly return to hypoxic levels (1%). Door is opened every ~24-48 hours to remove a sample. Gas levels have been manually checked to confirm incubator is at 1% O2.
Any reccomendations would be much appreciated! :)
Hi Devin,
Clearly I can't say anything for certain that the door being open for short periods of time is the cause of your issues but here are a few items to think about.
Door opening effect: When the incubator environment is disturbed, the cells can be affected. The frequency of disturbance can vary quite a bit from experiment to experiment and batch to batch and is a bit tough to fully grasp without controlling for it. While you are trying to mitigate the door open time it is still an unknown what is happening to your cells and the stress they are undergoing from being at 1% to something far greater than 1%.
Few other suggestions/ideas: As I have limited information I am assuming you are changing media from time to time. You should ensure you are preconditioning that media at 1% as well and not just taking it off the lab bench. I've seen this mistake made and its basically like taking your cells from 1% directly to room air instantly.
From my experience with standard incubators while they say something like 8 percent once you close the door they are often higher than that even when one is diligent in being quick with door opens. The reason for that is the systems are often binning a few measurements which keeps the levels looking a bit more stable. Beyond that, the oxygen sensor is very often located near the source of oxygen being purged into the system to show its responding quickly (keeping the number a bit lower than it is across the entire incubator). I wouldn't be surprised if most oxygen from the environment isn't cycled into your system in less than 20-30 seconds of door open time.
One last point is have you compared what you are getting as far as results to physoxic oxygen levels? I would say do this as opposed to comparing to benchtop/CO2 incubator results as these are very likely hyperoxic for your cells. It is a far better to have a control group to compare to that is physiologically normal than providing overly high oxygen values.
Use this review to help you get going and note section VI. REPLICATING NORMOXIA IN VITRO - 5 - C focuses entirely on the heart. This is a fantastic review otherwise and helps immensely with understanding physoxia and hypoxia!
Article Defining Physiological Normoxia for Improved Translation of ...
Workstation to remove doubt: I would say if this is something your lab is going to keep doing long term than it may be worth investing in something that removes this door opining issue. There are several companies that make systems that remove this variable including the one I'm with, Oxford Optronix. Few items worth considering if you go this route to help with reproducibility.
https://www.oxford-optronix.com/resources/the-reproducibility-issue-within-hypoxia-chambers-and-a-simple-solution-to-fix-it
Best of luck,