Live/Dead Cell Viability Assay

The viability of the cells was qualitatively evaluated to get an indication of whether fibroblasts survive and grow in the CHI/CNF hydrogel scaffolds. Cultivation of at least 6 days was possible and proliferation was observed. The cells were inspected for viability by fluorescent staining with a live/dead staining kit: calcein AM/ethidium-homodimer-1, LIVE/DEAD™ viability/cytotoxicity kit, (Thermo Fisher Scientific, Leicestershire, UK). After 1, 3, and 6 days of cell culture in the 3D hydrogel scaffolds as described above, a cell washing step was performed with Hank’s balanced salt solution (HBSS, Gibco, Thermo Fisher Scientific, Leicestershire, UK) and the freshly prepared LIVE/DEAD solution was added to each sample. Samples were kept at incubation conditions for 15 min. After staining, the wells were imaged using a confocal laser-scanning microscope (Leica TCS SPE, Wetzlar, Germany). As the LIVE indicator, calcein AM marks cell cytoplasm in green fluorescence; and as the DEAD indicator, ethidium homodimer-1 stains cell nucleus in red fluorescence.

Cell counting: Image analysis of the LIVE/DEAD cell viability assay micrographs was performed with Fiji (v1.52u), an ImageJ-based program [75]. Images were converted to 8-bit and the contrast was normalized via ‘histogram normalization’ command. The trainable weka segmentation tool was then used for cell segmentation [76]. Cells were counted using the Fiji “find maxima” command with a constant noise level to distinguish cell maxima vs. background noise. The results were exported to Excel for further statistical analyses.

Statistical analysis: All cell counting data were expressed as mean ± standard deviation (SD). Statistical analysis was performed by the one-way analysis of variance (ANOVA) using the software STATISTICA 10.0 (StatSoft Inc: Tulsa, OK, USA, 2011), followed by the Tukey’s HSD post hoc test if significant differences were found (p < 0.05) in the ANOVA.