Live dead solution

Cell-agarose constructs were divided into two halves, washed with 1× PBS and incubated in 700 μL live/dead solution (Thermo Fisher Scientific, Cat: L3224) for 18 min to stain live and dead cells using green fluorescent calcein-AM (4 mM) and red fluorescent ethidium-homodimer-1 (2 mM), respectively. Then, samples were washed with 1× PBS to remove the residual dye and imaged under fluorescence imaging using a Nikon TiE microscope with a DS-Qi2 camera (Nikon Instruments Inc., Melville, NY, USA). 10× and 4× cross-sectional images were taken of each gel with 10× images used for automatized quantification of cell viability (percentage live cells =s number of live cells over total cells) using MIPAR Image Analysis Software (Tang et al., 2019 (link)).

Cell-laden printed scaffolds (n = 3 per group) were washed with PBS and then immersed in the LIVE/DEAD solution (ThermoFisher, Waltham, MA, USA) for 15 min at 37 °C after 3 and 7 days of culture. After 15 min, the LIVE/DEAD solution was removed, and the samples were washed with PBS and stained with DAPI (Sigma, St. Louis, MO, USA) for 15 min at 37 °C. Scaffolds were washed with PBS once more and then visualized using a fluorescent microscope (Zeiss Axiovert) (Zeiss, Dublin, CA, USA) with Lumenera Infinity 3 (Teledyne, ON, Canada). Cell viability measurements were determined through ImageJ. The presence of cells within the cell-laden scaffolds were also confirmed with DAPI. Briefly, cell-laden scaffolds (n = 3 per group) were fixed with 10% formalin (Sigma, St. Louis, MO, USA) for 15 min and then washed with PBS. Afterwards, the scaffolds were placed into Tissue-Tek (Leica, Wetzlar, Germany) at −80 °C for 30 min and then sectioned at 20 µm using a cryomicrotome (Leica, Wetzlar, Germany). The cryosections were then stained with DAPI for 5 min at 37 °C and visualized with the fluorescent microscope.