Morphological analysis of OC-k3 cells under in vitro acoustic stimulation: integration of traditional and AI-based approaches

Sensorineural hearing loss is mainly caused by irreversible damage to the hair cells of the organ of Corti, structures that are highly sensitive to mechanical stimuli. Understanding how these cells respond to physical stress is essential to clarifying the mechanisms of vulnerability and adaptation. This study investigated the effects of low-frequency acoustic stimulation on the murine OC-k3 cell line to determine whether structural remodeling of the cytoskeleton and nucleus can serve as a reliable biomarker of cellular response to physical stress. Cells were exposed to controlled acoustic stimuli using a previously validated in vitro platform and were physically characterized with respect to substrate displacement and acceleration. Subsequently, the samples were analyzed by immunofluorescence and processed with an advanced image analysis pipeline to quantify global morphometric parameters and topological descriptors of the actin cytoskeleton. The data obtained were finally processed using supervised machine learning models to evaluate the predictive power of morphology with respect to stimulation conditions. The analysis revealed statistically significant alterations in cytoskeletal architecture, confirming a cellular response to the stimulus that is nevertheless predominantly local rather than global. Computational models did not identify linear frequency-dependent predictive patterns, indicating that intra-condition biological variability prevails over the effect of stimulation, confirming the inherently non-linear nature of the mechanosensitive response. Overall, the work demonstrates that although 2D static morphometry can detect vibration-induced local remodeling, it captures only a fraction of the biological complexity of the phenomenon. The results suggest the need to integrate dynamic or molecular metrics into future predictive models to fully decode cellular adaptation to acoustic stimuli.