For EB viability measurements, the tissues were removed from flow after 12 hours of perfusion. Next, the tissues and their molds were first immersed in a large bath of ice-cold PBS. The perfusion chambers were opened by unscrewing the front panel and removing the glass window. The tissues were then sectioned using a pair of microdissection scissors and transferred via aspiration into a 35-mm petri dish containing a cut silicone gasket to house the tissue for imaging. Next, the PBS was changed to mTeSR1 and PVA medium (4 mg/ml) by repeatedly flushing ~50% of the volume at a time with fresh mTeSR1. An equal volume of 2× concentrate LIVE/DEAD solution [calcein (1 μl/ml), ethidium homodimer-1 (EthD-1, 4 μl/ml), and Hoechst solution (0.5 μl/ml)] was then added, and the tissue was incubated at 37°C for 30 min. After incubation, LIVE/DEAD images were acquired on a confocal microscope (ZEISS) using a 5× ApoFluor objective. A cell number–normalized viability score was calculated as the ratio of the area of a thresholded calcein-AM viability signal to the area of a thresholded Hoescht signal. To obtain cell viability data for cardiac tissues, the same process was used. However, the tissues were incubated on ice immediately before imaging to suppress beating artifacts during imaging.

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