Live/Dead Cell Viability Assay

The evaluation of cell viability was performed at 7 and 14 days in vitro (DIV) using the Live/Dead assay (Molecular Probes). After rinsing hydrogels with PBS (3× for 5 min each) they were incubated at room temperature with PBS containing 2 μM Calcein-AM (stains the cytoplasm of viable cells green) and 4 μM ethidium homodimer-1 (stains the nuclei of dead cells red). After 40 min, the cell-laden hydrogels were rinsed in PBS and immediately imaged using a confocal microscope (Zeiss LSM 880 inverted confocal laser scanning microscope). Because the hydrogel area exceeded an individual confocal image field we acquired the complete hydrogel image using the tile module included in the Zen confocal software (Zeiss). Starting at the surface and imaging down toward the center of the hydrogel, the first and last z positions for each z-stack/tile were defined for a total thickness of 100 or 200 μm. The acquired image tiles were then combined into one final output by a stitching process using a 10% tile overlap. For automated cell counts, and estimation of cell viability, the stitched output images were imported to the 3D rendering software Imaris (Bitplane). We used the spot detection module, and spots were defined using cell count parameters for size and fluorescence strength of voxels to represent each cell soma. Parameters were then subjected to multiple image tests between manually counted and automated cell counts in multiple regions of interest of the imported tiled images to ensure accuracy. Because the quantification of spots using 200 and 100 μm thick tiled z-stacks did not differ statistically within hydrogels, we did the analysis using the thinner tiled z-stacks images. After validation, parameters were saved and then applied through the entire 100 μm z-stacks and overall cell count data was obtained for each image. Cell viability was calculated as (number of green stained cells/number of total cells) × 100%.