2.3.1. Cellular Cytotoxicity

Cytotoxicity test was performed according to the ISO 10993-5 standard. Cytotoxicities (for 3 and 5 days) for the produced sponge were performed on human fibroblast cells (HFFF2).

One of the methods for assessing cytotoxicity is to measure cell viability by MTT or (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. For the test, HFFF2 cells were seeded in 96-well plates containing an RPIMI medium supplemented with 10% FBS. Sample pieces were placed in the center of each well, and cells were incubated for different time points (24, 48, and 72 h). Untreated cells were considered as negative controls. After the incubation periods, cells (containing samples/control cells) were incubated with 150 μL fresh medium with 50 μL MTT solutions (2 mg/mL in PBS) for 4 h at 37 °C. Then, the MTT-encompassing medium was detached, and a mixture of 200 μL DMSO and 25 μL Sorenson’s glycine buffer (0.1 M glycine, 0.1 M NaCl, pH 10.5) was added to each well; the cells were incubated once more for 30 min at 37 °C. The wells without cells were considered blanks. The absorbance of plates was measured at 570 nm by a microplate reader. Cell viability was read as follows.