Cytotoxicity Evaluation of Nanomaterials

Rat NR8383 cells [60 (link), 61 (link)] were originally purchased from ATCC (USA) and cultured in F-12K medium supplemented with 2 mM glutamine, penicillin/streptomycin (100 U/10 mg/mL; and 15 % (v/v) fetal calf serum (FCS; all from PAN Biotech GmbH, Germany). Cells were grown in 500 mL flasks (Greiner, Germany) under standard cell culture conditions (37 °C; 5 % CO₂) and passaged once a week. For the in vitro tests, cells were detached from the substrate by mechanical agitation, dispersed by pipetting, seeded into 96-well plates at 3 × 10⁵ live cells per well and incubated in F-12K medium supplemented with 5 % FCS for 24 h. For test material application, supernatants were withdrawn, and test material-containing phenol red-free F-12K medium (Biochrom GmbH, Germany), supplemented with 2 mM glutamine and 100 U/100 μg/mL penicillin/streptomycin, was applied onto the cells. To correct for test material-specific adsorption and/or scattering of light, cell-free NM-containing controls were included in all test runs for all dilution steps. Cells were incubated with particles for 16 or 1.5 h. For the determination of LDH, GLU, and TNF-α release, cell culture supernatants were sampled after 16 h of incubation. In a parallel approach, supernatants were sampled after 1.5 h of incubation to assess H₂O₂ formation.