2.6.3. Live/Dead Assay

Briefly, thin hydrogels encapsulating hCSCs (2 × 10⁶ cells/mL) were prepared by placing a drop of hydrogel formulation (12 µL) at the center of a sterile coverslip. Then, another coverslip was placed immediately on top of the drop to yield a hydrogel sandwiched between the coverslips. The sandwiched hydrogel was then placed in culture media for a few minutes and then the top coverslip was gently released and pushed aside using a pair of sharp pointed forceps without damaging the underlying hydrogel. The hydrogels were cultured for a period of 5 days, following which they were incubated in calcein acetoxymethyl (calcein-AM, 0.2 µg/mL) and ethidium homodimer (2.5 µg/mL) (Invitrogen, Paisley, UK) for 15 min in supplemented DMEM at 37 °C to stain for live cells and dead cells. Live cells were visualized as green and dead cells as red under a fluorescence microscope (EVOS FL Auto2, Thermo Fisher Scientific, Waltham, MA, USA).