Mimicking hypoxic conditions in nonhypoxic geometries (1% O2 flush, DMOG, CoCl2)

To mimic hypoxic conditions in nonhypoxic geometries, a custom hypoxia chamber was designed using AutoCAD (Autodesk; San Rafael, CA) and machined at the Johns Hopkins University Whiting School of Engineering Machine Shop. This custom hypoxia chamber facilitated flow of mixed gases and real-time monitoring of O2 using commercially available O2 sensors (PreSens). In particular, 1% O2 (Airgas; Radnor, PA) was continually flushed through the chamber commencing immediately following ECFC encapsulation, and O2 was monitored along the culture period. Bright-field images were captured at predetermined time points to monitor cell morphology (Olympus IX50).

DMOG and CoCl2 act as PHD inhibitors, which up-regulate HIFs. Stock solutions of DMOG were made in diH2O [according to the manufacturer and previous literature (41)]. Stock solutions of CoCl2 were made fresh before each use in diH2O [according to the manufacturer and previous literature (43)]. DMOG (1 mM) or CoCl2 (200 μM) was co-encapsulated with ECFCs. Bright-field images were captured at predetermined time points to monitor cell morphology (Olympus IX50). ImageJ (public domain) was used to quantify cell clusters. Graphs were generated using GraphPad Prism 6 (GraphPad Software Inc., La Jolla, CA).

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