Live/Dead Cell Viability Assay

Live/Dead staining was performed using a commercially available kit (L3224, ThermoFisher). Staining solution was prepared fresh by addition of Calcein AM (0.5 μL/mL) and ethidium homodimer-1 (2 μL/mL) to pre-warmed PBS and wrapped in foil to prevent photobleaching. Bioprinted constructs cultured in expansion media for 1 or 7 days were washed with PBS (pre-warmed to 37 °C) and incubated in staining solution (0.5 mL, 30 min, 37 °C, wrapped in foil). After incubation, the samples were imaged using a widefield microscope (Calcein AM filter = GFP, ethidium homodimer-1 filter = Texas Red, Leica DMI6000 inverted epifluorescence microscope, equipped with a Leica LASX live cell imaging workstation and a Photometrics Prime 95B sCMOS camera). Cell counting was performed on widefield image stacks using FIJI.