3.13. In Vitro Cell Viability Assay by the MTT Method

L929 mouse fibroblasts and MRC-5 and HDFa human fibroblasts were cultured in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin in T-75 cell culture flasks. They were grown at 37 °C and 5% CO₂. The cell culture medium was changed every 48 h [83]. At 100% confluence, the cells were trypsinized (trypsin-EDTA) for viability analysis.

The effect of polyurethanes on cell viability was evaluated by the MTT method defined by ISO/CD 10993-5. Cells were seeded in 96-well plates at a concentration of 4.0 × 10⁴ cells per well in supplemented medium and cultured at 37 °C and 5% CO₂ for 24 h. Subsequently, PU cylinders of 3 mm × 2 mm (diameter × height) (previously sterilized under ultraviolet UV light (260 nm) for 30 min on each side [33]) were placed in 100 μL of supplemented medium. The materials were left in contact with the polymers for 24 h at 37 °C and 5% CO₂. The supernatant and polymers were then removed, and the MTT solution (12 mM in PBS) was added to a total volume of 100 μL and incubated for 4 h at 37 °C. The supernatant was removed, and 100 μL of dimethyl sulfoxide was added and incubated for 15 min at 37 °C. The optical density was then determined in a plate reader (Bio-Tek ELx800 Microplate Reader, Highland Park, Winooski, VT, USA) at 570 nm. A polypropylene biomaterial (PP) was used as a positive control for cell viability, and doxorubicin (DOXO) was used as a negative control. All tests were performed in triplicate. Cell viability was determined according to Equation (4):

where Abs_sample corresponds to the absorbance value of the cells after treatment with the PU, and Abs_control corresponds to the cells without treatment.