S 4800 2 sem

The organoids were analysed by brightfield and scanning electron microscopy (SEM). The samples were analysed on days 1, 7, 14, and 21 during differentiation. For SEM, the samples were cut to 1 mm diameter, fixed in 4% glutaraldehyde (Sigma-Aldrich), and then gradually dehydrated through an ethanol series, using sequentially higher concentrations of ethanol (70%, 80%, 90%, 95%, and 100%) for 10 min each. Finally, samples were further dehydrated in 50% hexamethyldisilazane (Sigma-Aldrich) diluted with ethanol for 10 min, followed by full immersion in absolute hexamethyldisilazane, and left to evaporate overnight in a fume cupboard. Control scaffold particles (no cells) were also incubated for 7 days in organoid medium, at 37°C under 5% CO₂ in air, before being fixed and dehydrated. SEM was carried out using a Hitachi S-4800 II SEM with an accelerating voltage of 1 kV at 110x and 5000x. Samples were mounted onto SEM stubs using a double-coated carbon conductive tape (Acros Organics, supplied by Fisher Scientific).
Cell viability was also assessed after 14 and 21 days using Live/Dead assay kit (Life Technologies, Paisley, UK), in which cells were stained with calcein acetoxymethyl (0.1 μg/ml) and propidium iodide (1 μg/ml) and viewed by fluorescence microscopy.

For scanning electron microscopy imaging, samples were fixed using 4% formalin and dehydrated in increasing ethanol concentrations prior to lyophilization. All samples were sputter-coated for 20 s and viewed using a Hitachi S-4800-II SEM. A Nikon AZ100 inverted light microscope was used for histological imaging.

Small gel pieces (2 × 5 mm) were fixed with 2.5% glutaraldehyde (pH 7.2) at 4 °C for 2 h, and subsequently washed three times in phosphate buffer (0.1 M, pH 7.2). The ethanol with different concentrations of 50%, 70%, 80%, 90%, and 100% was used to dehydrate, and each dehydration was maintained for 10 min. The gel pieces were then vacuum freeze-dried. Their microstructure was observed using an S-4800II SEM (Hitachi, Ltd., Tokyo, Japan). Digital images were obtained at an accelerating voltage of 10 kV and 2000× magnification [35 (link)].