Physiological Oxygen is Healthier for Cell Cultures

“Culturing cells in ambient air could be far from physiological with respect to oxygen. Oxygen is a surprisingly neglected factor [in cell culture].” Timpano and Uniacke

Drs. Timpano and Uniacke, HypOxystation users at University of Guelph in Ontario, have published a very thorough study examining the molecular basis of cells’ reactions to differing levels of hypoxia. In their paper “Human Cells Cultured Under Physiological Oxygen Utilize Two Cap-binding Proteins to Recruit Distinct mRNAs for Translation” (Journal of Biological Chemistry 291:20; 2016), they examine 2 different translation initiation proteins, eiF4E and eiF4E2, that are activated under either high (>8% O2) or low (<1% O2) oxygen levels, with the aid of mTORC1 or HIF-2α, respectively, and activated simultaneously in an area of low- to mid-level physioxia (1-8% O2). Timpano and Uniacke were able to stably and accurately create low oxygen in their HypOxystation by HypOxygen, which provides a closed workstation environment that enables researchers to culture and manipulate cells inside the chamber through gloveless sleeves, eliminating the negative consequences of spikes of higher oxygen and lower temperatures encountered in an incubator as cell cultures are growing. Their research into translational modulation of the proteome using the HypOxystation gives seminal insights into physioxia as the natural condition for cells, both in vitro and in vivo.

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Examining the Role of Autophagy in Hypoxic Tumors

HypOxystation users Tan et al. at the University of Toronto published a paper in June examining the significance of autophagy in cancer development (“Role of Autophagy as a Survival Mechanism for Hypoxic Cells in Tumors”, Neoplasia (2016) 18, 347–355). Autophagy as a means of recycling cell components is induced under stress conditions such as hypoxia, and Tan et al. investigated the correlation of hypoxia and autophagy in solid tumors in the context of resistance to cancer therapeutics. 

Cells were cultured in the H35 HypOxystation  for up to 48 hours at hypoxia (0.2 %) and compared to cells grown at ambient oxygen level. Gene silencing of autophagy proteins ATG7 and BECLIN1 with shRNA resulted in decreased cell survival under hypoxia, and inhibition of autophagy with pantoprazole exacerbated the loss of viability in the knock-down cells under hypoxia, demonstrating the cyto-protective effects of these autophagy proteins. Using the Seahorse XFe Analyzer to assess oxygen consumption in wild-type and silenced cells, Dr. Tan’s lab found reduced respiration when autophagy is disrupted, possibly due to accumulation of dysfunctional mitochondria in these mutant cells. The H35 HypOxystation  Dr. Tan’s lab used for these studies creates a closed environment with controlled temperature, humidity, CO2 and oxygen, in which cells are cultured and manipulated over the course of days and weeks without the need to transfer into ambient conditions, ever. The combination of HypOxystation and i2 Instrument Workstation is custom-designed to accommodate the specific requirements of the Seahorse XFe Analyzer for the duration of the metabolism assays investigating oxygen consumption and extracellular acidification.

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Examining the influence of hypoxia and normoxia on the expression of the H2S-producting enzymes CBS, CSE and MPS

Approximately 80% of renal cell carcinomas are of the clear cell subtype ccRCC, and common characteristics of this cancer include inactivation of the Von Hippel-Lindau tumor suppressor, leading to dysregulation of HIF-1 and 2α transcription factors and ultimately a pseudohypoxic tumor signature. Altered metabolism and upregulation of angiogenic factors result, and while therapies addressing individual such shifts are employed, an approach that targets the fundamental basis of the dysregulated survival and proliferation of ccRCC cells would increase effectivity while reducing systemic side effects.

HypOxystation users Sonke et al. in Canada examined the involvement of hydrogen sulfide H2S in angiogenesis, cytoprotection and metabolism in VHL-deficient ccRCC, where endogenous H2S accumulates as mitochondrial oxidation ceases under hypoxia and pseudo-hypoxia (“Inhibition of endogenous hydrogen sulfide production in clear-cell renal cell carcinoma cell lines and xenografts restricts their growth, survival and angiogenic potential; Nitric Oxide. 2015 Sep 15;49:26-39 “). The Sonke lab used the H85 HypOxystation by Don Whitley Scientific to examine the influence of hypoxia (1%) and normoxia on the expression of the H2S-producing enzymes CBS, CSE and MPS; cell viability under treatment with hydroxylamine HA, a CBS/CSE inhibitor, was also compared at normoxia and at hypoxia in the HypOxystation. The closed hypoxic environment created in the HypOxystation provides stable parameters favoring in vivo physiology of cancer cells. Using an avian chorioallantoic membrane xenograft system, they examined the inhibitory effects of H2S on neovascularization.

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H35 HypOxystation used in heart regeneration project

Dr Vaibhao Janbandhu is a Postdoctoral Research Scientist at the Victor Chang Cardiac Research Institute (VCCRI) in Sydney. He has been in contact with Don Whitley Scientific to explain how his lab’s work has benefited from the use of a Whitley H35 HypOxystation. Vaibhao uses the HypOxystation to isolate, culture and characterise adult cardiac stem cells (CSCs).

Dr Janbandhu had already been using a H35 that was set up at the institute for almost three years before he got his own unit installed last year. Specifically, his project is to find new ways to stimulate heart regeneration during ageing and after heart attack. For this he needs a way to isolate, culture and characterise adult CSCs. In Vaibhao’s words the H35 Hypoxystation seems well suited for this application: “the DWS HypOxystation provides a highly stabilised environment in which levels of oxygen, carbon dioxide, temperature and humidity are precisely controlled and it will be an integral part of the project to advance the project aims”.

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Treating advanced cancers by implanting polymer-entrapped palladium nano particles within a tumor

Selective activation of a drug precursor in an orthogonal manner can reduce the adverse systemic effects of the therapy. In a 2015 paper published in Scientific Reports (Vol 5, 9329), HypOxystation users Weiss et al. describe a therapeutic strategy to treat advanced cancers by implanting polymer-entrapped palladium nanoparticles within a tumor in order to continuously induce the activation of propargylated Pro-FUdR to FUdr, a DNA synthesis-disrupting antimetabolite, which is administered enterally. In their paper “Palladium-mediated dealkylation of N-propargyl-floxuridine as a bioorthogonal oxygen-independent prodrug strategy”, they describe the balancing act necessary to design and synthesize a compound that is inactive but resistant to metabolic degradation outside of the tumor, reducing its cytotoxicity for the patient 6,250-fold, which can then be chemically activated by palladium, aided by the specific micro­environment present inside the tumor.

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