Closed-loop dynamic negative pressure cell culture system for real-time quantitative analysis of fibroblast wound healing.

Negative pressure wound therapy (NPWT) is widely used to promote tissue repair. Its therapeutic efficacy is strongly dependent on the applied pressure mode and temporal profile. Despite increasing clinical interest in dynamic pressure regimens, the mechanobiological effects of dynamic negative pressure on cellular wound healing remain insufficiently characterized, primarily due to limited pressure controllability and insufficient real-time monitoring in existing experimental models. Here, we present a novel in vitro platform that enables precise and controllable application of dynamic negative pressure alongside continuous, quantitative assessment of cell migration. The system integrates closed-loop pressure control, a custom-designed airtight cell culture chamber suitable for long-term sterile culture, and real-time live-cell imaging. This configuration ensures stable delivery of continuous, intermittent, and dynamic pressure waveforms with minimal attenuation, thereby enabling accurate reproduction of prescribed mechanical stimuli. Using NIH/3T3 fibroblast scratch assays, scratch closure process was quantified under multiple negative pressure regimens. All negative pressure conditions significantly accelerated wound closure compared with controls. Notably, dynamic negative pressure involving cyclic oscillations between -75 and -125 mmHg not only enhances fibroblast migration but also promoting fibroblast activation and α-SMA-associated myofibroblast-like phenotypic changes, with a 2-min oscillatory cycle yielding the fastest wound closure. These findings demonstrate that temporal modulation of negative pressure enhances fibroblast migration and activation, highlighting the potential of dynamic NPWT for optimizing wound healing. The platform provides a robust, quantitative framework for studying NPWT-induced mechanobiology and systematically refining dynamic pressure regimens.