Samples from each test were cultured and tested for cell viability at 0 (D0), 1 (D1), 3 (D3) and 7 (D7) days after the bioprinting procedure. Cellular viability of each sample was analyzed using Invitrogen Live/Dead kit (Invitrogen™ R37601, Thermo Fisher Scientific, Waltham, MA, USA), staining live cells in green (488 nm) and dead cells in red (570 nm).

The cell viability images were obtained using a fluorescence microscope (Nikon TE2000-S, Tokyo, Japan) and cell viability was quantified using open-source ImageJ 1.52p software (National Institutes of Health, Bethesda, Rockville, MD, USA) by converting live and dead cells images of each test to 8-bit grayscales images, adjusting the histogram to get brighter images and reducing the background noise using the subtracting background process. Then, a composite image was obtained using the previous adjusted images. Finally, live and dead cells were manually counted to obtain the cell viability calculated as: live/(live + dead).

Cell viability results were processed using Graphpad Prism v. 9.1.0 (Graphpad Software, San Diego, CA, USA) using a two-way ANOVA with Tukey’s multiple comparison test, with 95% confidence interval (significance threshold p = 0.05).

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