It is quite known that it requires up to 24 hours exposures in hypoxic condition (1% O2) to establish acute hypoxia in cancer cells cultured in monolayer, whereas > 24 hours (2-5 days) have been shown to induce chronic hypoxia, more often through the hypoxia/reoxygenation cycles.
My question is, how do we establish this acute/chronic hypoxia in 3D cultures? As we know, it takes longer time to form spheroid, and after they reached a certain spheroid size, they also established a hypoxic region within the individual spheroids. In my case, I usually observe spheroid formation on starting day 4, with more compact morphology observed on day 7, and by the time they reached day 15, (I am using 6 wells Ultra-low attachment plate from Corning, seeding densitiy 5000 cells to enrich cancer stem cells).
If we are to culture these spheroids under hypoxic condition, what would be the ideal condition to mimic the acute/chronic hypoxia, in terms of culture duration? Anyone have any idea on any other consideration should be taken to establish acute/chronic hypoxia when working with tumourspheres/3D culture?
Any opinions/suggestions are greatly appreciated!!
Hello Ain.
Not easy to get a understandable principle, I guess. No one is convincingly demonstrating the differences between acute and chronic states of cells although claims vary, leading to confusion. And all cells and tissues also differ each other.
1) Determine a fixed target gene to look at the expression patterns in monolayer cell culture in a time-course manner under 1% O2, ie., to reduce parameters. At 5, 10, 20, 40 and 80 min for rapidly changing gene activation, and at 6, 12, 24h, 2d, 4d, and 8d hypoxia setting-up to get total RNAs from each dish if you can. Better to start -8d, -4d......6h dishes to get all groups together for simultaneous assay of fresh samples collected. Do similar way for 6~24h dishes, too. You need to control normoxia cell dishes for all the time points to compare target gene. It is critical to start very low cell concentration (a few hundreds) in 10mm dish, bearing 8d-culture period in your mind although you could negotiate cell numbers in early dishes to get enough RNA for RT-PCR. The results will provide you when your cells show hypoxic or acute gene expression patterns under the simplified parameters (1% O2 and free access of nutrients and gases to all cells).
2) In sphere culture, as you mentioned, outer cells are more free to get access to nutrients and gases in the medium while inner cells are not so, even leading to necrotic states (dying or dead). Therefore, it would be meaningless to determine hypoxic state in sphere culture. These sphere are so different from live tissues (in vivo), having no capillaries or other vascular systems in them. So, someone else's reports on organs or tissues may not be applicable to your spheres.
3) Protein analysis of monolayer and spheres will also provide further accurate determination of acute and chronic hypoxia at each time point. Comparing two cultures on protein levels (not RT-PCR this time, Western blot of the same target gene) would eliminate necrotic cells within the spheres.
As you already realized, the above-mentioned should be done before you jump into your aimed study with your sphere culture under acute and chronic hypoxia. Decision is yours.
Best wishes.
Acute hypoxia, which is in vivo caused by a temporary closure of vessels, cannot be reproduced in cell culters, neither 2D or 3D. What you can produce there is a general state of hypoxia. In 3D spheroids, this is usually in their center.
Best wishes
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