# Influence of Exposure Time and Driving Frequency on Cytotoxicity in In Vitro Ultrasound With Constant Mechanical Indices

## TL;DR

This study shows that in vitro ultrasound cytotoxicity depends on mechanical index, frequency, and exposure time, not just the mechanical index alone.

## Contribution

The study introduces new indices combining MI, frequency, and exposure time to quantify cell damage in vitro.

## Key findings

- Cell damage in vitro is influenced by mechanical index, ultrasound frequency, and exposure time.
- Cavitation and acoustic streaming are identified as the main causes of cell injury.
- Using MI alone is insufficient to predict cytotoxicity in vitro.

## Abstract

Sonochemistry has become increasingly important in bioengineering research, and many in vitro and in vivo bioapplications have been developed. Cytotoxicity is always a concern in its implementation. For in vivo treatments and studies, mechanical index (MI) is known to ensure biocompatibility, and even in vitro MI has been used. Because cell characteristics and acoustic phenomena differ in vitro and in vivo, we questioned using MI in vitro. The in vitro cytotoxicity of ultrasound exposure should be investigated to support the development of cutting‐edge sonochemistry. In this study, a system for irradiating cultured cells with 1–2 MHz‐range ultrasound was developed to demonstrate the invalidity of employing MI alone in vitro. The results showed that cell damage is defined by the MI, ultrasound frequency, and exposure time, which are new indices for quantifying cell damage. Furthermore, cavitation and acoustic streaming are shown to be the main scientific factors that injure cells.