Encapsulated Cell Viability Assay

Encapsulated cell viability was assessed on days 1, 7 and 21 by calcein acetoxymethyl ester/ethidium homodimer fluorescent stain as described previously.1 (link) Briefly, hydrogels were submerged in media, 2 mM calcein acetoxymethyl ester, and 4 mM ethidium homodimer (LIVE/DEAD^® Viability/Cytotoxicity Kit for mammalian cells; Life Technologies, Grand Island, NY). Viable cells within the hydrogel sheets were identified based on intracellular enzymatic conversion of non-fluorescent calcein-AM to green fluorescent calcein while ethidium homodimer-1 penetrated damaged membranes of dead cells and upon binding to nucleic acids fluoresced bright red. After 10 min in a tissue culture incubator, each hydrogel was imaged under an epifluorescent microscope (Zeiss Axiovert Observer Z1) and 10–15 optical slices at 28 µm intervals were taken in the z-plane at three diagonal positions (e.g., top left, center, and bottom right) in the x–y coordinates. Green fluorescent images for live cells and red fluorescent images for dead cells for each optical slice were separately processed on ImageJ software (Rasband, National Institutes of Health) to obtain cell counts.