Abstract:In this study, the preparation method and primary culture conditions of Chlamys farreri gill cells were optimized. The toxic effects of benzo[a]pyrene (B[α]P) were compared and analyzed by three cell activity detection techniques. The results showed no significant difference in the survival rates of the primary cultured gill cells disinfected with 1% penicillin-streptomycin solution (Pen-Strep) and gentamicin for 10~30 min. There were also no bacterial infections observed in the microscopic examinations after 30 min, and the cells were in good condition. The effect of trypsin digestion time on the harvest of gill cells was significant. Within 15~25 min of digestion, the survival rate of the gill cells was higher, and the best trypsin digestion time was 25 min. With 150~300 g relative centrifugal force, the morphology and survival rate of the gill cells significantly differed, and the survival rate and cell integrity were better at 150 g. There were no changes in survival within 6~12 h after adding fetal bovine serum (FBS, 5%~20%). At 24 h, the survival rates of the 5% and 20% treatment groups decreased significantly, but the 10% and 15% treatment groups were unaffected. The cytotoxicity of B[α]P on the scallop gill cells was detected by three cell activity tests, and the results showed no change in the activity of the gill cells with the trypan blue exclusion assay. The cell counting kit-8 assay showed that the activity of the gill cells was significantly inhibited at the highest concentration (16 μg/mL), while the neutral red assay showed a positive toxicity correlation between the B[α]P concentration and time. These results suggest that the best preparation method for C. farreri gill cells is disinfection for 30 min, trypsin digestion for 25 min, relative centrifugal force at 150 g, and the addition of 10% fetal bovine serum to the primary culture medium. The neutral red assay can be used as a sensitive index to evaluate the toxicity of B[α]P.
