Cell viability was tested using the Live/Dead Assay Kit (Thermo Fisher, catalog no. L3224) containing calcein-AM and ethidium homodimer dyes for staining live and dead cells, respectively, as previously described (34). Spheroids were formed from HES3 CMs and incubated at 37°C and 5% CO2 for 5 days with CDM3 medium changed every other day. Hoechst 33342 (Thermo Fisher, catalog no. 62249), calcein-AM, and ethidium homodimer dyes were added with a final concentration of 1 μg/ml, 2 μM, and 4 μM, respectively, to each sample and incubated for 30 min at 37°C and 5% CO2. The spheroids were then treated with 20 μM blebbistatin (Sigma-Aldrich, catalog no. B0560) to decouple excitation and contraction, leading to inhibition of spontaneous cell beating (31). The spheroids were washed with Tyrode’s solution, and one spheroid was encapsulated by 3D-SR-BA. The live-cell imaging was performed at 37°C using upright confocal microscope (Nikon A1R) under 20×/0.50 numerical aperture (NA) water immersion objective, with perfusion of Tyrode’s solution with 4 μM ethidium homodimer dye at 37°C. The spheroid encapsulated by 3D-SR-BA alongside with the non-encapsulated control was imaged immediately after encapsulation and every 30 min after the encapsulation up to 3 hours. The procedure was repeated three times for different sets of spheroids.

% Viability quantification was evaluated as previously described (34) using the following formula%Viability=Total cells (blue)Dead cells (red)Total cells (blue)×100where blue refers to the cells stained by Hoechst and red refers to the dead cells stained by ethidium homodimer. Total cell count and dead cell counts were determined by counting the Hoechst- and ethidium homodimer–stained nuclei across the spheroid images for each of the encapsulated spheroid and non-encapsulated control spheroid separately (n = 3 each), using the spot tracking algorithm in Imaris software (Imaris 8.2 Image Visualization and Analysis, Bitplane, Oxford Instruments).

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