Evaluation of the location and probability of acoustic cavitation in healthy and cancerous 3D cell cultures

• Cavitation occurred exclusively in extracellular space across 40 3D cell cultures. • Cavitation thresholds consistently decreased with increasing ultrasound pulse length. • Healthy rat muscle cells had lower cavitation threshold than cancerous counterparts. • Cell presence reduced cavitation thresholds compared to cell free scaffolds. • AFM revealed cancerous cells were stiffer than their healthy counterparts. Acoustic cavitation, or the rapid growth and collapse of pre-existing bubble nuclei under a varying acoustic pressure field, plays a critical role in the safety and efficacy of biomedical ultrasound. However, the availability and behavior of bubble nuclei, which act as seeds for acoustic cavitation, remain largely unexplored in biological tissues. In this study we evaluated the spatial location of bubble nuclei and cavitation thresholds in biological systems by culturing four rat-derived healthy and cancerous cell lines (musculoskeletal L-6 and L-8; hepatic BRL-3A, and McA-RH7777) in two commercial 3D scaffolds: PureCol EZ-Gel and Cultrex Ultimatrix. A single element 3.68 MHz focused ultrasound transducer (f#=1) induced low-density cavitation with pulse durations of 10–300 μs at 1 Hz PRF and peak negative pressures up to p- = 6.07 ± 0.13 MPa. The spatial locations of bubbles were monitored and captured using brightfield microscopy and high-speed photography (20,000 fps). Bubble formation was observed exclusively in the extracellular scaffold, with no intracellular cavitation detected in any of the 40 cell culture samples. In 3D cell cultures of healthy musculoskeletal cells, lower cavitation thresholds (∼4.1 MPa) were observed compared to their cancerous counterparts (∼4.9 MPa; p < 0.001) for 200 μs pulses, possibly due to the ECM-modifying behavior of L-8; however, the trend did not extend to liver cells where cavitation thresholds were similar. Increasing pulse length consistently reduced cavitation thresholds across all cell-scaffold combinations. Together, these results suggest that cavitation is predominantly extracellular, which may have implications for the safety and efficacy of emerging therapeutic ultrasound applications.