Quantitative study on cytotoxicity of methylene blue to human nucleus pulposus cells by cell counting-kit 8 assay / 中国组织工程研究

ABSTRACT

BACKGROUND: Our preliminary study has shown that methylene blue exerts toxic effect on human nucleus pulposus cells in a concentration-dependent manner, but the toxic range remains unclear. OBJECTIVE: To determine the critical range of the cytotoxicity of methylene blue to nucleus pulposus cells by cell counting-kit 8 (CCK-8) assay. METHODS: The nucleus pulposus was from a patient with intervertebral disc herniation. The nucleus pulposus cells were extracted and cultured. Passage 1 cells mere used to make cell suspensions. The cells were divided into nine groups for culture: blank control (only the medium, CCK-8 solution), control (only medium, cells and CCK-8 solution), and methylene blue groups (1%, 0.5%, 0.1%, 0.05%, 0.01% and 0.005% of methylene blue). The absorbance values were measured by CCK-8 assay at 24, 48, 72 and 96 hours after incubation. The cell viability was calculated, and the color was observed. RESULTS AND CONCLUSION: The color in the control group was the darkest, and the color in the methylene blue groups became lighter with the concentration of methylene blue increasing, and the 0.05%, 0.01%, 0.005% and 0.001% methylene blue groups showed darker color similar to the control group. The absorbance values in the 0.1% methylene blue group were significantly less than those in the control group (P＜ 0.05). There were no significant differences in the absorbance values and cell viability between 1% and 0.5% methylene blue groups (P ＞0.05). The absorbance values and cell viability in the 0.1% methylene blue group were significantly higher than those in the 0.5% methylene blue group, but were significantly less than those in the 0.05% methylene blue group (P ＜ 0.05). Thus, methylene blue exerts cytotoxicity to human nucleus pulposus cells, and the critical value of toxicity is between 0.1% and 0.05% and close to 0.05%. However, the exact value needs a further investigation.