4.6. Cytotoxicity Studies

Cytotoxicity of the polymerized hydrogels were assessed using the MTT assay. Hep G2 cells were cultured in Dubecco’s modified eagle medium (DMEM) supplemented with 10% fetal bovine serum, penicillin/streptomycin and L-glutamine at 37 °C with 5 % CO₂. For toxicity testing, three different concentrations (0.2, 0.6, 1.0 mg/mL) of each hydrogel formulation were dissolved in media. Hep G2 cells were seeded at 1 × 10⁴ cells per 100 µL of media in 96-well plates and incubated in an atmosphere of 5 % CO₂ at 37 °C. Once the desired confluency was obtained, the cells were washed with PBS buffer and treated with 100 µL of the different hydrogel concentrations for 24 h at 37 °C with 5 % CO₂ atmosphere. A 0.5 mg/mL MTT solution was prepared by dissolving 50 mg of MTT salt in 10 mL of sterile PBS buffer and then filter sterilized. This solution was further diluted in media (1:10 dilution) and used for the assay. Once the test media was removed, cells were incubated with the 0.5 mg/mL MTT solution for 4 h to form formazan crystals. After this, the solution was removed and 100 µL of di methyl sulfoxide (DMSO) was added and the resulting solution was measured for absorbance at 570 nm using a microplate reader. Results are presented as percentage cell viability.