After SWIFT printing of cardiac tissues in machined perfusion chambers, the tissue was capped with hcECM to provide mechanical stability required to maintain patency for long-term culture. The mold was then transferred to a 37°C, 5% CO2 incubator for 45 min to complete gelation of the OBB-ECM matrix, and to melt the printed sacrificial gelatin filaments and gelatin that surrounds the tissue. After incubation, silicone molds were sealed via an excess of autoclaved SE1700 silicone base (without cross-linker) into the gap at the top of the mold, while machined perfusion chambers were sealed by screwing on the polycarbonate lid. The sealed perfusion devices were then transferred to a custom-made tissue perfusion housing that held the tissue vertically and adjacent to a media reservoir, containing mTeSR1 media with PVA (4 mg/ml; for EB perfusion) or CMM (for cardiac tissue perfusion) with 1% antibiotic-antimycotic (Thermo Fisher Scientific). Next, the tissue was maintained at 37°C, where the sacrificial gelatin ink is molten, and a peristaltic pump (Ismatec Inc.) was used to introduce fluid media that removes the gelatin and leaves behind open channels. For the cardiac tissue perfusion, after evacuating the printed gelatin at a vascular flow rate of 0.01 ml/min for 5 min, the inlet for the external perfusion was opened, and the molten gelatin that surrounds the cardiac tissue was evacuated at a high perfusion rate (0.8 ml/min) for 5 min. To ensure the removal of all gelatin, this process was repeated but with a vascular perfusion rate of 0.02 ml/min. Over the course of ~1 hour, the perfusion rate was then increased gradually to its final perfusion rate. Perfusion rates used are 250 μl/min for Fig. 3 (C and D), 100 μl/min for EB-tissue perfusion for viability measurements, and 500 μl/min for cardiac tissue perfusion. For specific experiments, the media reservoir was hyperoxygenated by means of bubbling a mixture of sterile-filtered 95% O2/5% CO2 (Airgas Inc.) through the media at a volumetric flow rate of ~10 ml/min.

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