Low-intensity focused ultrasound enables temporal modulation of human midbrain organoid differentiation

Abstract

Controlling the precise timing of biosignaling cues in complex 3D models such as organoids is critical for guiding cellular differentiation and functional maturation. Ultrasound stimulation, a next-generation neuromodulation modality, offers unique advantages due to its target specificity, ability to elicit mechanosensitive responses, and high spatial resolution. However, there is still a lack of precision stimulation platforms and comprehensive biomarker assays for selective control of organoid development. Here, we introduce a modular piezoelectric ultrasound stimulation platform that integrates seamlessly with conventional multi-well plates, enabling selective neuromodulation of midbrain organoids (mBOs). We demonstrate that ultrasound can specifically modulate cellular differentiation by promoting dopaminergic progenitor markers while delaying terminal differentiation. Importantly, ultrasound stimulation did not induce cellular damage, as confirmed by the absence of apoptosis and DNA damage markers. This work demonstrates the potential of focused ultrasound as a safe, non-invasive, and tunable biophysical cue for temporal regulation of organoid differentiation and maturation. Significance StatementOrganoids are three dimensional in vitro models derived from human tissue that recapitulate the complex features of organs. However, precise modulation of organoid differentiation relies on biochemical factors, which are limited in their temporal controllability. In this study, we demonstrate that low-intensity ultrasound stimulation enables temporal modulation of midbrain organoid differentiation using a modular multi-well stimulation platform. In particular, we show that ultrasound promotes the proliferation of dopaminergic progenitor cells. These findings suggest that ultrasound can serve as a supplementary mechanical cue to regulate midbrain organoid development.