Here we propose simple methods to induce hypoxia in cell cultures and simple tests to evaluate the hypoxic status of the cultures.
Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells. This decrease of oxygen tension can be due to a reduced supply in oxygen (causes include insufficient blood vessel network, defective blood vessel, and anemia) or to an increased consumption of oxygen relative to the supply (caused by a sudden higher cell proliferation rate). Hypoxia can be physiologic or pathologic such as in solid cancers 1-3, rheumatoid arthritis, atherosclerosis etc… Each tissues and cells have a different ability to adapt to this new condition. During hypoxia, hypoxia inducible factor alpha (HIF) is stabilized and regulates various genes such as those involved in angiogenesis or transport of oxygen 4. The stabilization of this protein is a hallmark of hypoxia, therefore detecting HIF is routinely used to screen for hypoxia 5-7.
In this article, we propose two simple methods to induce hypoxia in mammalian cell cultures and simple tests to evaluate the hypoxic status of these cells.
Cell proliferation and viability in hypoxic conditions vary a lot depending on the cell types. Therefore, you should adjust the cell number or the number of cultures plates you start with to make sure you will have enough cells/proteins for your experiments.
The Cobalt Chloride method has the advantage to be inexpensive and fast. This product mimics hypoxia by inducing HIF-1/3α but can also regulate other genes, make sure this product is adapted to your project by checking what other effects it could have on the function and phenotype of your particular cells independently of the “mimic-hypoxic” effect. Another drug also used to mimic hypoxia is Deferoxamine mesylate (DFO, used at 100 μM final concentration). The use of these drugs allows the experimentator to open the culture plate/dish/flask many times without affecting the “hypoxic condition”.
The level of oxygen contained in the gas mixture can vary depending on your experiments and cell types as hypoxia values vary depending on the tissues and cell types. Indeed, some cells are hypoxic in 5% O2 other need less than 1% O2 to be hypoxic. 5%CO2 is added to the gas mixture to stabilize the pH of the cultures, the rest of the gas is usually Nitrogen.
Hypoxic chambers have the advantage of not using drugs that can alternate cell behavior independently of the oxygen tension. However, not all types of experimentation can be done as oxygen re-enters the chamber at each opening and thus lessens hypoxia. You should consider this factor in your experiments; hypoxia/re-oxygenation is a specific condition that can affect some cell types. An alternative is to use hypoxia workstations (connected to pre-mixed gas tanks or to gas mixing systems) or larger hypoxia incubator (hypoxia processing chamber with glove box) that allows the experimentation to change media and manipulate cells in continuous hypoxic environment. Various hypoxic chambers have been commercialized over the past decade and should be chosen according to your laboratory used, projects, space, size, and budget. Always ensure the good use and condition of your chamber to guarantee correct culture conditions. In general when using a chamber, the level of hypoxia can be modulated by selecting the various gas mixes (i.e. 1%, 5% 10% oxygen).
Regarding the detection of HIF-1α, it is important to know that some cancer cell line express HIF-1α in normoxia. It is therefore critical to use a normoxia-control to determine the basal level of HIF in these cell lines. HIF-1α can also be present in normoxia in non-malignant cells following cell stimulation or stress. This could also occur if these cells are starved, make sure your cell cultures are properly feed and maintained.
The authors have nothing to disclose.
Name of the reagent | Company | Catalogue number |
---|---|---|
Anti-HIF-1α | Invitrogen | 458400 |
Cobalt (II) Chloride | Sigma | C8661-25G |
Incubator Chamber | Billups-rothenberg | MIC-101 |
HRE-Luciferase plasmid | Adgene | Plasmid 26731 |