Cytotoxicity Evaluation of Hydrogels

In order to evaluate the cytotoxicity of the tested hydrogels on HaCaT and BJ cells, the Neutral Red Uptake Assay (Sigma Aldrich, Poznan, Poland) was used. This test was performed based on the procedure described previously [67 (link)]. After 2 and 24 h of exposure to extracts of the six GEN-loaded hydrogels, cells were incubated for 2 h with a neutral red dye (40 µg/mL), which was dissolved in serum-free DMEM medium. After incubation with NR, cells were washed with sterile PBS and 150 µL of decolorizing buffer (C₂H₅OH/CH₃COOH/H₂O, 50%/1%/49%) was added to each well to release cellular dye into the PBS solution. After shaking the cells for 15 min the absorbance of the dissolved dye at λ = 540 nm was determined using a FilterMax F5 Multi-Mode microplate reader (Thermo Fisher). The mean absorbance of the control cells (untreated with the extracts) was taken as 100% cell viability and used to calculate the percentage of viable cells in the experimental samples treated with the test hydrogels. As a part of the study, three independent experiments were carried out, in which each concentration of extracts was tested using four replicates.