A Novel Chip for Cyclic Stretch and Intermittent Hypoxia Cell Exposures Mimicking Obstructive Sleep Apnea

Abstract

Intermittent hypoxia (IH), a hallmark of obstructive sleep apnea (OSA), plays a critical role in the pathogenesis of OSA-associated morbidities, especially in the cardiovascular and respiratory systems. Oxidative stress and inflammation induced by IH are suggested as main contributors of end-organ dysfunction in OSA patients and animal models. Since the molecular mechanisms underlying these in vivo pathological responses remain poorly understood, implementation of experimental in vitro cell-based systems capable of inducing high-frequency IH would be highly desirable. Here, we describe the design, fabrication, and validation of a versatile chip for subjecting cultured cells to fast changes in gas partial pressure and to cyclic stretch. The chip is fabricated with polydimethylsiloxane (PDMS) and consists of a cylindrical well-covered by a thin membrane. Cells cultured on top of the membrane can be subjected to fast changes in oxygen concentration (equilibrium time ~6 s). Moreover, cells can be subjected to cyclic stretch at cardiac or respiratory frequencies independently or simultaneously. Rat bone marrow-derived mesenchymal stem cells (MSCs) exposed to IH mimicking OSA and cyclic stretch at cardiac frequencies revealed that hypoxia-inducible factor 1α (HIF-1α) expression was increased in response to both stimuli. Thus, the chip provides a versatile tool for the study of cellular responses to cyclical hypoxia and stretch.

Intermittent hypoxia increases kidney tumor vascularization in a murine model of sleep apnea

“…To assess the potential sources of VEGF under IH conditions, 5x10 4 RENCA cells, 15x10 4 RAW 264.7 cells and 6x10 4 HAEC cells were exposed to IH (cycles alternating 20% O 2 −1% O 2 every 30 s) or continuous RA (21% O 2 ) as previously described [23]. After 24 h, total RNA from RENCA, RAW 264.7 and HAEC cell cultures were extracted using the RNeasy kit (Qiagen, Hilden, Germany).…”

Technical Feasibility and Physiological Relevance of Hypoxic Cell Culture Models

“…Unlike in other microfluidic settings, cells are not in the chamber itself but rather are in direct contact with it by growing on a gas-permeable membrane, which allows for close control and fast equilibration of pericellular O 2 , while diminishing mechanical stress. Furthermore, the modulation of pressure in the chamber mediates a cyclical stretching of the cell culture that simulates the periodic expansion of the heart or lung (Figure 2D) (63). Using a similar principle, the development of a PDMS pillar with an oxygen perfusion channel, coated with Parylene-C to ensure better oxygen isolation and faster equilibration, has been reported.…”

“…Additionally, the meniscus of the medium forming within the culture well-causes a significant difference in the diffusion distance across the culture surface, which requires maintaining the cells in the middle of the well (62), which is difficult to achieve when using cell lines with significant proliferative capacity. Alternatively, researchers used stirring or shaking of the culture vessel as a simple method to increase oxygen diffusion (36), although this was at the cost of inducing mechanical stress to the cells (63).…”

“…A venting tube connected to the well-leads to a solenoid valve (not shown), which serves the purpose of periodically changing pressure inside the well, applying indirect mechanical stimuli to cells growing on the outside of a gas-permeable membrane of the well. Adapted according to (63) under the CC BY license. (E) Cell culture insert for intermittent hypoxia.…”

“…To this end, a microfluidic chip, capable of creating pO 2 gradients and evoking multiple oxygenation states, has been developed (116). A myriad of other microfluidic devices could also be devised for the purpose of accurately recreating tumor phenotypes in a dish (63,117).…”