A microfluidic oxygen gradient demonstrates differential activation of the hypoxia-regulated transcription factors HIF-1α and HIF-2α

Abstract

Gas-perfused microchannels generated a linear oxygen gradient via diffusion across a 100 μm polydimethylsiloxane (PDMS) membrane. The device enabled exposure of a single monolayer of cells sharing culture media to a heterogeneous oxygen landscape, thus reflecting the oxygen gradients found at the microscale in the physiological setting and allowing for the real-time exchange of paracrine factors and metabolites between cells exposed to varying oxygen levels. By tuning the distance between two gas supply channels, the slope of the oxygen gradient was controlled. We studied the hypoxic activation of the transcription factors HIF-1α and HIF-2α in human endothelial cells within a spatial linear gradient of oxygen. Quantification of the nuclear to cytosolic ratio of HIF immunofluorescent staining demonstrated that the threshold for HIF-1α activation was below 2.5% O2 while HIF-2α was activated throughout the entire linear gradient. We show for the first time HIF-2α is subject to hyproxya, hypoxia by proxy, wherein hypoxic cells activate HIF in close proximity normoxic cells. These results underscore the differences between HIF-1α and HIF-2α regulation and suggest that a microfluidic oxygen gradient is a novel tool for identifying distinct hypoxic signaling activation and interactions between differentially oxygenated cells.

Bioengineered Systems and Designer Matrices That Recapitulate the Intestinal Stem Cell Niche

“…Oxygen gradients with arbitrary profiles (eg, linear, exponential, and nonmonotonic) have been generated as stable or reconfigurable gradients. 140 , 141 Oxygen gradients compatible with an open-well culture also have permitted direct observation of cell behavior in response to varying oxygen levels. 142 Vertical oxygen gradients over Caco-2 cells established by simultaneous perfusion of oxygenated and deoxygenated media through 2 separated microchannels permitted co-culture of Caco-2 cells with an obligate anaerobe under conditions representative of the gastrointestinal human–microbe interface.…”

Engineering the Cellular Microenvironment of Post-infarct Myocardium on a Chip

“…Cardiac myoblasts near the hypoxic end of the gradient exhibited altered morphology, including reduced cell area and actin disintegration, which was accompanied by mitochondrial dysfunction and decreased cell viability ( 59 ). In addition to chemical methods ( 60 – 63 ), microfluidic devices have also been developed to generate physical oxygen gradients by culturing cells on a gas-permeable membrane above microchannels for gas flow ( 64 – 66 ). However, these methods have not been extensively applied to cardiac cell types.…”

Metabolic Switching of Tumor Cells under Hypoxic Conditions in a Tumor-on-a-chip Model

“…Alternatively, polymer films with low oxygen permeability were embedded in PDMS devices to induce hypoxic conditions on 2D [30] or 3D cell cultures [31]. In a different approach, and by taking advantage of the gas permeability of PDMS or similar silicone materials, dedicated channels perfused with pre-conditioned fluids were placed on the top [15,[32][33][34] or next to culture chambers [35,36], allowing a quick switch of the oxygen tension in a 3D tumor model [37], to create oxygen gradients in cellular models [38,39] or to simply supply enough oxygen to hepatocytes [15]. Along a similar line, solutions supplemented with an oxygen-scavenging chemical were utilized to create hypoxic conditions [40] or oxygen gradients [41][42][43][44][45], in combination in one report with a thick oxygen-impermeable material (PMMA or polymethylmethacrylate) sheet placed on the top of the device [46].…”