Induction and Testing of Hypoxia in Cell Culture

Abstract

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][2][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][6][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. Video LinkThe video . This product is soluble in water (100 mg/ml), yielding a clear, red solution.1. Prepare a 25mM stock solution in sterile dd water, (prepare immediately before use) 2. Use CoCl 2 at the final concentration of 100μM in your regular cell culture media to induce hypoxia. 3. Add the CoCl 2 containing media to your cells and incubate the cultures for 24hours in a conventional incubator (37°C; 5% C0 2 ).The above concentration works for the cell lines we have tested but each cell line should be tested at various concentrations (to establish a dose-dependent curve) as well as at various incubation times in order to limit drug related toxicity and optimize the assay. 3. Place the cell culture in the hypoxic chamber. Also place a Petri dish containing sterile water in the chamber to provide adequate humidification of the cultures. 4. Place the "twin" cell culture in normoxia as control. 5. Make sure your trays are secured and not moving, and then close the chamber with its lid. Correctly position the steel ring clamp to ensure the hermetical closure of the chamber and close it. 6. To create hypoxia, attach the tubing to a "hypoxia tank" containing a 1% O 2 gas mixture. If you have a flow meter connected to your tank your chamber will be directly connected to it (gas tank-flow meter -chamber). We use a flow meter incorporated in our regulator. 7. It is important to remove most if not all oxygen present in the chamber and in your media, to do so flush the chamber by opening the gas tank at a flow rate of 20 liters per minutes for 7-10 minutes; then quickly turn off the gas flow and completely close the chamber by closing both white clamps. 8. Return the chamber to a conventional incubator for the desired period of time. If you use large cultures, allow media in cultures to de-gas for 1-2 hours and then repeat flush. Hypoxia induced in Modular Incubator Chamber

Chloroquine Exerts Anti-metastatic Activities Under Hypoxic Conditions in Cholangiocarcinoma Cells

“…We demonstrated that CoCl 2 -induced hypoxia significantly increased metastases in both CCA cell lines, using a transwell migration assay. We also confirmed that CoCl 2 truly induces hypoxic conditions as demonstrated by the suppressing the protein level of PHD-2 and the increased level of hypoxia responsive elements HIF-1α and VEGF (Wu and Yotnda, 2011). In addition, we revealed that CoCl 2 -induced hypoxia facilitates CCA cells to metastasize by altering the level of proteins involved in EMT-mediated metastasis, i.e., reduction of E-cadherin to N-cadherin ratio, a hallmark of EMT (Gravdal et al, 2007;Chen et al, 2014) were observed for M139 and M214 cells.…”

“…Under hypoxic conditions, the PHDs process is inhibited, theSuyanee Thongchot 1,3 , Watcharin Loilome 1,3 , Puangrat Yongvanit 1,3 , Hasaya Dokduang 1,3 , Raynoo Thanan 1,3 , Anchalee Techasen 2,3 , Nisana Namwat 1,3 * angiogenesis, and intracellular proteins which allow cells to survive in O 2 deprivation by reprogramming their metabolism (Semenza, 2012). Cobalt chloride (CoCl 2 ) is a chemically induced hypoxic-mimicking condition which occurs by inhibiting PHD and stabilizing HIF-1α, and it is frequently used for inducing a hypoxic condition in many in vitro systems (Yuan et al, 2003;Wu and Yotnda, 2011). It has been showed that 100 μM of CoCl 2 treatments to human prostate cancer cell lines resulted in elevated VEGF mRNA and protein secretion, and drove cells to be highly invasive metastatic phenotype (Liu et al, 1999).…”

Visualization of Calcium Dynamics in Kidney Proximal Tubules

“…3 In an attempt to mimic ischemia (hypoxia) and reperfusion (reoxygenation) in isolated renal PTE cells, we have pretreated the cells in media containing 200 mM CoCl 2 , reported to cause hypoxic conditions in various cell types in vitro. 18,19 After 24 hours' pretreatment, exchange of the media for oxygenated media containing no CoCl 2 resulted in a large transient increase in cellular calcium levels, while addition of ATP still produced a rapid calcium rise ( Figure 2D). The exchange of media without CoCl 2 pretreatment did not alter intracellular calcium levels (Supplemental Figure 2F).…”

The Hypoxia-Reoxygenation Injury Model

“…Chemical hypoxia models that use mitochondrial uncoupling agents or respiration blockers reproduce the rapid onset of ischemia but there is no way to study the recovery processes that occur during reperfusion [6][7][8][9]. The significance of true hypoxia-reoxygenation models is in the capacity for recovery from the hypoxic phase, which makes these models especially useful for ischemia-related experiments.…”