Study on key parameters of the in vitro cytotoxicity test in nanomaterials assessment

Objective: To investigate the key factors and parameters of the in vitro cytotoxicity test(MTT assay and LDH assay) for assessing nanomaterials referring to ASTM,so that to provide evidences for establishing standards of nanomaterial’s cytotoxicity test.Methods: The detection wavelength of MTT assay and LDH assay were obtained by full-wavelength scanning;the concentration and storage condition of the positive control APAP were tested;the experimental factors of MTT assay were tested by changing the MTT media with or without glycine buffer after adding DMSO;the experimental conditions of LDH assay were measured by changing the concentration of serum(10%,5%,2.5%) in cell culture media.The cytotoxicity of the silver nanoparticle containing hydrogel was assessed by refined MTT assay and LDH assay.Results: The double-wavelength was found at 540-570 /680 nm and 490 /680 nm,which is suitable for MTT assay and LDH assay respectively,in this experimental system.The proper concentration of positive control APAP was 20-30 mmol.L-1,which should be stored at-20 ℃ and will be stable for 14 days.The serum contained in the cell culture media could affect the LDH test with a serum concentration-depended manner,but this kind of interference could be deducted by the blank control.The IC50 of silver nanoparticle-containing hydrogel tested by refined MTT assay and LDH assay were 26.8 mg.mL-1 and 61.8 mg.mL-1,respectively.Conclusions: Two assessment points can be obtained in one experiment by combining re-fined MTT assay and LDH assay,so that the cytotoxicity of nanomaterials can be evaluated more comprehensively.In addition,this study defines the key factors and parameters of the cytotoxicity test(MTT assay and LDH assay) for assessing nanomaterials,and improves the feasibility of the method.