2.4.2. Cytocompatibility Assays

The cytocompatibility tests were performed on mouse fibroblast cells NIH/3T3 (ATCC ® CRL-1658 ™) in accordance with ISO 10993-5:2009 [57]. The protocol was refined in previous studies [58,59] and can be briefly summarised as follows:

Cell culture preparation: When cells reached confluence, they were detached with trypsin, collected and centrifuged at 200 × *g* for 10 min after trypsin inactivation with soybean trypsin inhibitor. After centrifugation, the cells were re-suspended, and their number was adjusted to 10<sup>5</sup> cells/mL. On each sample, 10<sup>4</sup> cells were seeded in 100 μL medium. The plates were inserted into the incubator for cell adhesion. After 4 h, 400 μL of the Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM-F12) (Sigma Aldrich, St. Louis, MO, USA) culture medium supplemented with 10% foetal bovine serum, was added. After 24 h, 50 μL of medium was collected to investigate the cytotoxic e ffect by the lactate dehydrogenase (LDH) assay (Thermo Scientific, Waltham, MA, USA).

Cell morphology: Epifluorescence microscopy was used to assess the morphology of 3T3 cells on the surface of the SBG samples. After 24 h of culture, the cells were fixed with 4% paraformaldehyde in phosphate bu ffered saline (PBS) for 15 min and then washed three times for 5 min with PBS. The cells were then incubated for 1 h at RT with 100 μL Phalloidin-AlexaFluor546 (Invitrogen, Carlsbad, CA, USA). After a series of three PBS washes of 10 min each, the cells were incubated with 1 μg/mL 4 , 6-diamidino-2-phenylindole (DAPI) (Sigma Aldrich, St. Louis, MO, USA). After a final washing step in PBS (two times for 10 min) and double distilled water (one time), the cells were imaged in a standard fluorescence microscope Leica DM6 B apparatus (Wetzlar, Germany), equipped with a Leica DFC 9000 GT camera and appropriate fluorescence objectives and filters.

Cell proliferation: The cellular proliferation was evaluated using the MTS (3-(4, 5-dimethyl thiazol-2-yl) 5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) kit (Promega Corporation, Madison, WI, USA). At the time of culturing, three wells were used to investigate the mitochondrial activity at T0 moment. Three hours after seeding, the cells were analysed under an inverted microscope to observe their adhesion. The well was filled with 400 mL of fresh complete cell culture medium and then 80 μL MTS bu ffer was added. After 1 h in the incubator, 120 μL of medium was transferred to 96-wells plates and the absorbance was read at 490 nm using a Zenyth 3100 multimodal microplate reader (Anthos Labtec Instruments, Salzburg, Austria). The optical density (OD) at the T0 seeding moment was calculated, and then was used to quantify the cell proliferation after 24 h.

Cell death: The cytotoxicity of SBG films was investigated using an LDH assay kit. After 24 h of cell culturing, 50 μL of the supernatant medium was collected and transferred into a 96-well microplate. An amount of 50 μL LDH substrate solution, prepared according to the manufacturer's instructions was added to each well. After 30 min of incubation, the reaction was stopped with 50 μL of stop bu ffer. The absorbance values at 490 and 620 nm were read with the Zenyth 3100 apparatus and the result was considered as the di fference between the absorption at 490 nm and that at 620 nm (taken as a standard). The cut-o ff value was obtained in the presence of the fresh culture medium.
