Development and characterization of a reproducible, low-cost in vitro ultrasound stimulation (LIVUS) method

Ultrasound has been used for decades in diagnostic and therapeutic applications. Low-intensity pulsed ultrasound (LIPUS) has recently emerged as a promising modality for low-energy therapies that rely on cell mechanotransduction rather than thermal effects. Despite the increased application of LIPUS for several applications in the clinic, including bone healing and cellular modulation, the translation of its applications requires reliable in vitro systems that enable systematic screening of ultrasound parameters and biological responses. However, existing in vitro stimulation platforms often lack thorough characterization of acoustic parameters or rely on complex, in-house devices that are difficult to reproduce. Here, we present a low-cost in vitro ultrasound stimulation (LIVUS) system based on standard cell culture consumables, including conventional plates and inserts. The LIVUS system provides acoustic transparency to stimulate cultured human umbilical artery smooth muscle cells (HUASMCs) and substantially attenuates unwanted acoustic reflections. Using the LIVUS system, we demonstrate that LIPUS induces mechanotransducive phenotypic modulation in HUASMCs. Our approach provides a practical and reproducible tool for investigating the effects of LIPUS on the phenotype of adherent cells.