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% O followed by 1 min at 2% O resulted in Po values ranging from 124 to 44 mmHg. Extending hypoxic and normoxic phases to 10 min allowed Po variations from 120 to 25 mmHg. The volume of culture medium or the presence of cells only modestly affected the Po 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.

Nonmuscle Myosin Light Chain Kinase: A Key Player in Intermittent Hypoxia‐Induced Vascular Alterations

“…After 3 days, the Transwells were placed in gas‐permeable 24‐well plates (Zell‐kontakt, Nörten‐Hardenberg, Germany) and preincubated for 30 minutes in culture medium with 5 μmol/L ML‐7, a selective MLCK inhibitor (Sigma‐Aldrich), diluted in dimethyl sulfoxide at a final concentration of 0.12% dimethyl sulfoxide in the well or with 0.12% dimethyl sulfoxide as vehicle. They were then exposed to 8 hours of normoxia (constant 16% O 2 and 5% CO 2 ) or to IH consisting of 5 minutes at 16% and 5 minutes at 2% O 2 with constant 5% CO 2 , as described 33. Oxygen concentration in the cell environment (ie, inside the Transwell) was monitored with an oxygen probe (Oxford Optronics, Abingdon, UK) and was found to cycle between 70 and 125 mm Hg.…”

“…They were then exposed to 8 hours of normoxia (constant 16% O 2 and 5% CO 2 ) or to IH consisting of 5 minutes at 16% and 5 minutes at 2% O 2 with constant 5% CO 2 , as described. 33 Oxygen concentration in the cell environment (ie, inside the Transwell) was monitored with an oxygen probe (Oxford Optronics, Abingdon, UK) and was found to cycle between 70 and 125 mm Hg. Transendothelial electrical resistance was measured before and after exposure to normoxia or IH with an epithelial voltometer (EVOM-2; World Precision Instruments).…”

Technical Feasibility and Physiological Relevance of Hypoxic Cell Culture Models

“…To overcome the above-mentioned limitations, placement of a commercially available culture dish with a gas-permeable bottom made of a fluorocarbon membrane in a modular incubator chamber (an air-tight sealed plastic chamber) filled with atmosphere-containing predetermined O 2 and CO 2 levels has been employed (45,64). Using this setup, adherent cells receive O 2 directly from the modular incubator chamber atmosphere via the permeable membrane without having to rely on diffusion through the medium, which has been shown to be both effective and simple in terms of being able to regulate pericellular pO 2 closely with relatively fast equilibration times (45,65).…”

“…The required gas mixtures are prepared and pre-warmed in a gas blender, brought into the plate holder by gas inlet tubing, allowing for unlimited diffusion of oxygen between the plate holder and adherent cells, and subsequently flushed out through a gas outlet tube to make room for a fresh gas mixture. Adapted with permission according to (64). O 2 levels, e.g., by a programmable digital controller, enables for rapid and reproducible exposure of cells to intermittent hypoxia, without the need for diffusion through a culture medium ( Figure 3A) (45,65).…”

“…The limitations of such an approach include the large volume of gases (O 2 , N 2 , and CO 2 ) required to achieve the rapid exchange of the inner cabinet atmosphere associated with significant culture media evaporation (despite humidification) and gas pressure changes inside the cabinet or inside the sealed culture dishes due to heat expansion of gas (45), all of which adversely affect the performance of IH exposure. Minoves et al (64) modified the setup by combining the gas permeable culture dishes with a customized plate-holder equipped with its own gas tubing. This was designed to seal the plate off from the surrounding atmosphere, replacing the hypoxic chamber with a significantly smaller space, and thus limiting the volume of air to be pumped in and out during each cycle (Figure 3B).…”