Real-time imaging and automated quantification of wound closure dynamics

To begin with, temporal changes in wound closure (%) under different FBS concentrations (0%, 5%, and 10%) were compared (Figure 2). With identical initial wound areas, wound closure was delayed in the 0% FBS condition, indicating reduced recovery capacity. In contrast, wound closure progressed rapidly in the 10% FBS condition, consistent with enhanced cell migration and proliferation (Figure 2A). The 5% FBS condition also showed closure, but at a slower rate than the 10% FBS condition. Quantitative analysis confirmed that the 10% FBS condition exhibited the steepest increase over time, whereas the 0% FBS condition showed only a slight change, highlighting that growth factor content in the medium significantly affects cell motility and wound closure dynamics (Figure 2B).

In addition to serum conditions, wound closure can be influenced by cell migration, proliferation, and cell death. To examine how perturbing these processes alters closure kinetics, additional wound-healing assays were performed with staurosporine, cytochalasin B, and doxorubicin at sublethal concentrations.

In representative images (Figure 3A), the 10% FBS control condition showed robust wound closure. In contrast, staurosporine, a potent apoptosis inducer2 , markedly reduced wound-healing activity. Cells displayed progressive morphological changes, including shrinkage, consistent with apoptosis (Figure 3A-B). Cell death became evident around 20 hours, causing the wound closure curve to plateau, with little to no further closure observed thereafter (Figure 3C).

Cytochalasin B also markedly reduced wound closure. Because cytochalasin B disrupts actin polymerization and impairs the cytoskeletal dynamics required for motility3 , this effect is consistent with reduced migratory capacity. No evident cell death was observed, but proliferation appeared suppressed (Figure 3B), limiting advancement of the leading edge and slowing closure (Figure 3C).

Interestingly, doxorubicin produced a wound closure rate similar to the control. As a chemotherapeutic agent that induces DNA damage4 , doxorubicin did not cause pronounced cell death under the conditions tested but appeared to suppress cell proliferation. Despite this growth inhibition, wound closure remained comparable to the control because cells showed extensive spreading while maintaining migratory activity. This suggests that suppressing proliferation alone may not be sufficient to reduce wound closure and that effective inhibition requires direct impairment of migration. Notably, under mild cytotoxic stress, doxorubicin may promote a compensatory response in which cells increase their footprint and continue collective migration despite reduced growth5 . These results demonstrate that wound closure curves vary markedly depending on the drug applied and can be categorized into distinct patterns (e.g., steep upward, gradual upward, or flat) based on their trends.

Taken together, these findings highlight the value of live-cell imaging for interpreting wound closure dynamics, as migration- and proliferation-associated behaviors can contribute differently depending on the condition or treatment. Distinguishing the relative contributions of these processes enables more accurate assessment of wound-healing activity and provides clearer insight into how external stimuli modulate collective cell behavior.

Conclusion

This application note highlights the effectiveness of the Celloger® Pro system for wound-healing assays. Using NIH-3T3 cells, the system clearly detected differences in wound closure rates across FBS concentrations and enabled classification of distinct drug-induced closure patterns. By automating both image acquisition and wound area quantification, the system enables high-resolution, time-dependent monitoring of wound closure with minimal manual intervention.

Notably, the advanced wound detection algorithm provides accurate and consistent segmentation of the wound region, even in low-contrast or partially closed gaps. This improvement enhances the reliability of quantitative analysis and reduces user-dependent variability. Overall, the ability to clearly distinguish wound closure responses under different conditions underscores the value of Celloger® Pro as a powerful all-in-one solution for regenerative medicine research and cell motility studies.