# Versatile High-Throughput Platform for Focused Ultrasound In Vitro Application
## 5 Conclusions  
In this work, we implemented and characterized two versions (LIFU and HIFU) of cell sonication devices that enable standardization of in vitro workflows for HTP investigations by parallelized sonication. The wide intensity and frequency bandwidths allowed an effective exploration of the fundamental mechanisms of the action of therapeutic ultrasound on a wide variety of in vitro models in preclinical research. In conclusion, different cell sonication tools optimized for 96-well plates were developed. The LIFU and HIFU versions have different advantages and limitations. The manufacturing process of the HIFU applicator based on individual transducers allows a preselection prior to integration, which, in the end, allows a higher yield and homogeneity. Furthermore, the implementation of a water-cooling system ensures that the sonicated samples are not subjected to heating by the transducer surface, thereby preventing any adverse effects. In addition, the cooling allows driving the transducers at duty cycles up to 100%. However, the risk of air bubbles trapped below the membrane is a side effect of water cooling, which must be considered. Finally, the limitation of sonication to only 32 of the 96 wells in parallel, which resulted in the use of focusing transducers with a footprint larger than the well-to-well distance, is a notable drawback of this setup. On the other hand, the main advantage of the LIFU variant is the capability for dry coupling, eliminating the risk of air bubbles and making the use more convenient. Providing access to all 96 wells furthermore makes it easier to achieve statistical significance when investigating therapeutic effects in vitro. Future work could focus on a combination of the two versions. For instance, a system with individually manufactured transducers integrated into standard housings would allow the preselection of a highly homogeneous subset of transducers and the integration of arbitrary transducer combinations (e.g., of different frequencies) in the same multi-well applicator. Such screwable and interchangeable transducers would not only increase flexibility but improve maintenance in case of transducer defects. In addition, ultrasonic transducers could be equipped with a customized acoustic lens to optimize focusing in the wells and to adapt the focus spot size better to the well geometry (or to some inserts, e.g., when using membrane models for BBB opening). Finally, it would be beneficial to equip the transducer elements with temperature sensors to facilitate temperature-controlled application and, thereby, ensure that the observed effects actually result from an acoustic effect in the cell medium and not from thermal diffusion. The potential for future integration of electronics with 96 transmission channels could also be explored. This could enhance the individualization of the sound output in each well, allowing for precise control over frequency, intensity, and transmission signal sequences. In a recent study, the potential of our cell sonication setup for investigation of tumor spheroids treatment was demonstrated [26]. The researchers showed that low-intensity pulsed-focused ultrasound reduced spheroid growth metabolic activity and increased DNA double-strand breaks in in vitro cancer cell models. Our future work will aim at developing multichannel electronics and adapted ultrasonic transducers to meet the great need for technologies for investigating the mode of action of ultrasound on biological cells and cellular networks as pre-studies for more efficient application of ultrasound in future therapies. Conceptualization, S.H.T. ; methodology, S.H.T., C.D., F.-J.B. and P.W. ; software, S.H.T. and W.B. ; validation, S.H.T., M.F., F.-J.B., W.B., L.L. and A.M.; formal analysis, S.H.T., M.F., W.B. and S.T.-M.; investigation, S.H.T., M.F., C.D., F.-J.B., P.W., W.B., L.L. and A.M.; resources, S.H.T. and A.M.; data curation, S.H.T., M.F., W.B., L.L. and S.T.-M.; writing--original draft preparation, S.H.T.