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

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 wide range of tunable waveform parameters, including pulse repetition frequency (PRF) and acoustic intensity (I). 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 parameter-controlled neuromodulation of midbrain organoids (mBOs). Using two distinct ultrasound regimes, 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.