Focused ultrasound (FUS) is a non-invasive technique producing a variety of biological effects by either thermal or mechanical mechanisms of ultrasound interaction with the targeted tissue. FUS could bring benefits, e.g., tumour sensitisation, immune stimulation, and targeted drug delivery, but investigation of FUS effects at the cellular level is still missing. New techniques are commonly tested in vitro on two-dimensional (2D) monolayer cancer cell culture models. The 3D tumour model—spheroid—is mainly utilised to mimic solid tumours from an architectural standpoint. It is a promising method to simulate the characteristics of tumours in vitro and their various responses to therapeutic alternatives. This study aimed to evaluate the effects of FUS on human prostate and glioblastoma cancer tumour spheroids in vitro. The experimental follow-up enclosed the measurements of spheroid integrity and growth kinetics, DNA damage, and cellular metabolic activity by measuring intracellular ATP content in the spheroids. Our results showed that pulsed FUS treatment induced molecular effects in 3D tumour models. With the disruption of the spheroid integrity, we observed an increase in DNA double-strand breaks, leading to damage in the cancer cells depending on the cancer cell type.

For the more efficient application of ultrasound in future therapies, fundamental research is needed on the mode of action of ultrasound on biological systems using therapeutic frequencies. To address this need, a new versatile high-throughput platform for focused ultrasound in vitro application was designed, developed, and characterized. The applicator was aligned with the dimensions of a 96-well plate and frequencies commonly used in the therapeutic ultrasound range (0.5–2.0 MHz). Two different platform configurations were developed: (a) a low-intensity version with 96 individual transducers allowing dry coupling of the well plate; and (b) a high-intensity version with water cooling, supporting parallel sonication of 32 out of 96 wells. The platforms were characterized by performing an analysis of the homogeneity of the sound pressure and intensity, the impact of filled volume per well, the cross-coupling effect between the wells, and the influence of the well plate. The low-intensity design delivers pressure levels up to 605 kPa inside the well with maximum I_SPPA values between 0.78 and 12.38 W/cm². In contrast, the high-intensity system achieves pressures up to 1460 kPa and a maximum I_SPPA of 72 W/cm² inside the wells. The successfully developed high-throughput platform supports parallelized sonication in standard, well-plate formats and is suitable for focused ultrasound applications in vitro.