Entrapped cells were liberated from scaffolds through enzymatic and mechanical digestion. Scaffolds were washed with PBS, followed by perfusion of 12 ml of trypsin for 5 min at 37°C. Trypsin was retrieved from the bioreactors and added to an equal volume of medium. Scaffolds were removed from bioreactors, minced, and immersed in trypsin for an additional 5 min at 37°C in a tube rotator (Thermo Fisher Scientific, Santa Clara, CA) at 15 rpm. The immunophenotype of retrieved cells was determined using flow cytometry. Briefly, nonspecific binding was blocked by incubation in Protein Block (AB156024, Abcam, Cambridge, MA) for 20 min and then incubated with antibodies against CD31 (303110), CD45 (368518), CD34 (343623), CD90 (328112), and CD73 (344008) (all from BioLegend, San Diego, CA) per the manufacturer’s instructions.

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