DescriptionBackground: Top2b is known to be essential in neural development. The lack of Top2b in embryonic stem cell (ESC)-derived neurons may cause premature cell death, as well as abnormal retinal development. So far, few research has focused on using fibroblasts to investigate the function of Top2b. Therefore, function of Top2b in fibroblast migration still remains unclear. Material and methods: Fibroblasts were obtained from postnatal day 1 (P1) mice and cultured for 7 days before transferred to microfluidic devices. Top2b inhibitor ICRF-193 was used to suppress Top2b function and determine the effect of Top2b on fibroblast migration. Fibroblasts were seeded on one side of two-chamber microfluidic devices and treated with 0.1 μM, 1 μM, and 10 μM ICRF-193, whereas equal concentration of DMSO and DMEM were used as controls. Continuous 7-day observation starts from 1 day after seeding cells on devices. Overnight time-lapse microscopy was performed on cells between 2 days and 4 days. Results: Significant differences in the number and speed of fibroblast cell migration were observed with ICRF-193 treatment. Higher concentration of ICRF-193 resulted in fewer cells in the migration chamber and slower migration speed. Conclusions: These results demonstrate an important role of Top2b in fibroblast migration, thus further making Top2b as a potential target for wound healing and treatment of cancer.