APS 2017 – Scientific Paper – Intermittent Hypoxia Model

University: Université Grenoble Alpes
Authors: Mélanie Minoves, Jessica Morand, Frédéric Perriot, Morgane Chatard, Brigitte Gonthier, Emeline Lemarié, Jean-Baptiste Menut, Jan Polak, Jean-Louis Pépin, Diane Godin-Ribuot and Anne Briançon-Marjollet
Journal: American Physiological Society 2017
Impact Factor: 3.22 (What is the IF?).
Application Note related to our instrument: Intermittent Hypoxia in Cell Culture

TITLE: An innovative intermittent hypoxia model for cell cultures allowing fast Po2 oscillations with minimal gas consumption.

ABSTRACT: Performing hypoxia-reoxygenation cycles in cell culture with a cycle duration accurately reflecting what occurs in obstructive sleep apnea (OSA) patients is a difficult but crucial technical challenge. Our goal was to develop a novel device to expose multiple cell culture dishes to intermittent hypoxia (IH) cycles relevant to OSA with limited gas consumption. With gas flows as low as 200 ml/min, our combination of plate holders with gas-permeable cultureware generates rapid normoxia-hypoxia cycles. Cycles alternating 1 min at 20% O₂ followed by 1 min at 2% O₂ resulted in Po₂ values ranging from 124 to 44 mmHg. Extending hypoxic and normoxic phases to 10 min allowed Po₂ variations from 120 to 25 mmHg. The volume of culture medium or the presence of cells only modestly affected the Po₂ variations. In contrast, the nadir of the hypoxia phase increased when measured at different heights above the membrane. We validated the physiological relevance of this model by showing that hypoxia inducible factor-1α expression was significantly increased by IH exposure in human aortic endothelial cells, murine breast carcinoma (4T1) cells as well as in a blood-brain barrier model (2.5-, 1.5-, and 6-fold increases, respectively). In conclusion, we have established a new device to perform rapid intermittent hypoxia cycles in cell cultures, with minimal gas consumption and the possibility to expose several culture dishes simultaneously. This device will allow functional studies of the consequences of IH and deciphering of the molecular biology of IH at the cellular level using oxygen cycles that are clinically relevant to OSA.

Intermittent hypoxia (IH) is the hallmark of obstructive sleep apnea (OSA), a common chronic disease affecting 5–20% of the general population and characterized by recurrent collapses of the upper airway, leading to the repetitive occurrence of oxygen desaturation/reoxygenation sequences. OSA is recognized as an important and independent risk factor for hypertension, coronary heart disease, and stroke and could also be associated with mild cognitive dysfunction. Moreover, recent studies suggest that the excess mortality in OSA could be at least partly due to an increased risk of cancer. The molecular pathways underlying the deleterious consequences of OSA are under investigation but cellular and molecular mechanisms remain poorly understood. Moreover, clinical research is limited by confounding factors that make it difficult to distinguish between the respective effects of intermittent hypoxia and comorbidities. Since stroke and coronary heart disease are common OSA-associated comorbidities, increased knowledge of the effects of IH exposure of endothelial cells and blood brain barrier appears to be crucial. Finally, IH is also observed in tumors and exposure of tumor microenvironment to IH might promote tumor growth and metastatic activity.

In this context, significant efforts were recently made in several leading laboratories in the field to obtain relevant models of IH in cell cultures. The achievement of rapid oxygen cycles in standard culture dishes is flawed by the very slow oxygen diffusion in culture medium in the absence of mixing. Moreover, thermally induced convective mixing of the media is not sufficient to ensure rapid oxygen equilibration across the height of medium thus limiting the development of relevant IH systems. For instance, one of the first cellular IH models, based on air flushing in a Lucite chamber, generated cycles alternating 15 s of 1% O₂ and 3 min of 21% O₂ that allowed only limited (between 50 and 70 mmHg) fluctuations in Po₂ in the culture medium. This highlights the challenge of oxygen diffusion for effective cell exposure to IH. Longer cycles have allowed cycling between 2% O₂ and 15% O₂, but with cycle durations of 1 h and 1.5 h, that are not clinically relevant in the context of OSA-related IH. Another strategy has consisted in using preequilibrated culture medium. In these systems, the use of preconditioned medium does ensure instantaneous oxygen changes at the cell level. However, repeated changes in medium complicate the measurement of soluble factor secretion and can induce an important shear stress that could impact cell activity and metabolism. More recently, a team has proposed a system in which gas was directly injected in the culture flask. They alternated 5 min of 16% O₂ and 5 min with 0% O₂, resulting in six cycles per hour, which was the best compromise to achieve sufficient oxygen variation amplitude.

Finally, the last type of system described in the literature uses gas-permeable dishes to obtain rapid and accurate cycles at the cell level. In this system, variations between 16% and 1% O₂ lead to similar oxygen variations in the culture medium within minutes, allowing a frequency of six cycles per hour without any change in medium or bubbling. The major disadvantage of this elegant setting is the high gas consumption necessary to replace the air volume of the cabinet incubator hosting the dishes. Achievement of the control normoxic exposure also relies on expensive premixed gas bottles (16% O₂, 5% CO₂, and 79% N₂). Recently, a variant of this system, based on air circulation underneath highly-permeable polydimethylsiloxane (PDMS) membranes for cell culture, achieved very fast oxygen cycles at the cell level in a system adapted for direct microscopy imaging. The major limitation of this model relies on its small size (4-mm-diameter dishes) and thus in the low number of exposed cells, preventing large-scale studies or the collection of cells for biochemical or molecular biology studies.

Therefore, our objective was to set up a cost-effective and rapid-cycling model producing IH cycles able to mimic the tissue oxygenation characteristics of OSA. We aimed at developing and characterizing a device that would allow rapid oxygen cycling and, minimal gas consumption, avoiding the use of expensive premixed gas bottles, and allowing exposure of multiwell plates and larger culture plates. The originality of this system is that it generates rapid cycles in culture media while minimizing gas consumption by using gas-permeable cell cultureware and custom-made holders in which the air is renewed only below the dishes.

FULL AND ORIGINAL SCIENTIFIC PAPER: Methods in Cell Physiology.

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