# Versatile High-Throughput Platform for Focused Ultrasound In Vitro Application
## 1 Introduction  
Ultrasound (US) has emerged as a prominent non-invasive therapeutic modality over recent years. There are 166 clinical indications that are being studied and treated with focused ultrasound (FUS) systems, which are in various stages of development [1]. Applications range from tumor therapy [2, 3, 4], neurodegenerative [5, 6] and psychiatric diseases [7], movement disorders [8, 9], pain [10], and stroke treatment [11, 12] to Alzheimer's disease [13]. For therapy of the above-named conditions, different ultrasound effects have been investigated, ranging from thermal ablation, mechanical fragmentation, (mild) hyperthermia, blood-brain barrier (BBB) opening, or targeted drug delivery (TDD) to ultrasound neuromodulation. A common point in all these applications is that theultrasound parameters, in particular, the frequency, the pressure, and the intensity, have a great impact on the resulting effect. In processes in which no irreversible effect on tissues is desired, such as BBB-opening, TDD or neuromodulation, the pressure and intensity have to be carefully adjusted such that there is no thermal or mechanical damage to the cells. On the other hand, ideal sonication parameters leading to the desired effect (e.g., transient BBB opening) with a high level of certainty are sometimes still unclear and need further investigation. Accordingly, to expand the field of application of FUS, new ultrasound therapy systems must be developed, and parameters that ensure the best therapeutic effect and outcome must be identified. While clinical translation is already ongoing in some fields, further pre-clinical work is needed in many ultrasound applications. For this purpose, in vitro studies are the preferred method of choice [14, 15], especially at an early stage of research and when statistical significance is sought, which can be achieved by parallelized cell model experiments more easily than on small animal models. However, there is a lack of ultrasound systems optimized for research into FUS effects on cell models which allows for a variation in crucial sonication parameters (e.g., sound pressure, frequency, modulation sequence). Current setups mostly rely on single-element transducers optimized for a defined spectral and pressure range, which are not adapted for high-throughput (HTP) cell culture workflows. In the past, setups for in vitro treatment have often been based on single transducer systems that were immersed, e.g., in a petri dish [16, 17]. However, this direct contact with the cell medium is a major disadvantage in terms of contamination and influence on the cell models. It requires intensive cleaning and sterilization efforts before and between studies and, furthermore, alters the cell environment even without actually applying ultrasound (just by immersing transducers in the medium). Further systems have been described in which water is used as a coupling medium and where a single FUS transducer is positioned in a bath below the cell medium. Such systems have been used for sonication in standard multi-well plates [18, 19], customized bioreactors [20], or single-cell culture dishes [21]. However, since only one US transducer is available, even when using a multi-well setup, only one experiment can be performed at a time, and statistical significance is hard to achieve. Multi-element US arrays initially designed and developed for clinical settings, in which the ultrasound focus is applied to the individual wells from the bottom of a water bath [22], have also been investigated. Further approaches are based on several (up to 5) US single-element transducers, which need to be mechanically maneuvered in the water bath [23]. Although it has been demonstrated that several wells of multi-well plates can be addressed by such systems, the very high costs [22] and the complexity [23] prevent the use of the routine tool in the research on in vitro models. Furthermore, standing waves [24] or undesired cross-coupling from well to well [25] can have an unwanted effect.