3.9.1. Cytotoxicity Assay

The in vitro cytocompatibility of scaffolds was tested following EN ISO 10993-5 standard protocols (n = 3). Samples were irradiated with UV-C (λ = 254 nm, 5 min, 80 W) for decontamination. Then, 1.5 mL of complete cell culture medium (DMEM 4.5 g/L D-glucose supplemented with 5% FBS, 1 mM L-glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin) was added to the scaffolds and kept at 37 ◦C under constant stirring for 72 h. Next, 0.1 mL of this extracting medium was added to murine fibroblasts L929 P32 previously seeded at 1.10<sup>4</sup> cells per well in a 96-well plate the night before to allow their adhesion. After 24 h of incubation at 37 ◦C in a humid environment, cell viability was assessed based on ATP quantification using CellTiter Glo (Promega G7571) according to the supplier instructions. Briefly, 50 μL of media was removed from each well, then 50 μL of CellTiter Glo reagen<sup>t</sup> was immediately added to each well containing the cells and incubated for 10 min in the dark. Finally, luminescence was read and the results for each sample were compared to TCPS. Negative (high-density polyethylene film) and positive (polyurethane containing 0.1% zinc diethyldithiocarbamate) controls were also tested.
