2.11. Cell Viability Assays

To test the polymer’s cell viability, a CellTiter-Glo^® Luminescent Cell Viability Assay (Promega Corporation, Madison, WI, USA) was performed, according to the manufacturer’s protocol. The assay was performed using the L929 mouse fibroblast cell line. Cells were seeded in a white 96-well plate (Greiner Bio-One) at 8500 cells per well. After 24 h of incubation, a dilution series from 3.9 µg mL⁻¹ to 500 µg mL⁻¹ of the polymer was prepared in RPMI 1640 (+10% FCS), and 100 µL of each concentration was added to the wells. Subsequently, the cells were incubated for further 24 h. After one day of incubation, 100 µL of the CellTiter-Glo^® reagent was added, following 2 min of shaking at 450 rpm and 10 min of incubation without shaking. Luminescence was determined with the Tecan Spark 20M (Tecan Group AG). A thiomersal 0.02% solution (Caelo, Hilden, Germany) served as a positive control, and untreated cells were set as 100% control. RPMI 1640 supplemented with 10% FCS served as the blank value and was subtracted from all the measured values. The final cell viability was calculated by setting the measured values in correlation to the untreated control. Cell viability values below 70% were defined as toxic, according to DIN EN ISO 10993-5 [36]. This assay was repeated twice with eight technical replicates, and data are shown as percentages (mean ± SEM).