In-vitro 3D organoid cultures constitute an essential component of modern-day biological research, with key application areas in drug and therapy testing, and Organ-on-a-Chip models. The increasing demand for reliable models comes with the challenge of boosting the throughput of production of these cultures. The currently researched 3D culture methods pose the challenge of not being contact-free, of limited versatility and throughput in organoid culturing, and often lack a stimulus to promote cell agglomeration. This work presents acoustofluidics as a stimulus-driven solution to promote the clustering of cells in a levitated, suspended environment. In order to establish this objective experimentally, an SBAW resonator was designed, fabricated, and tested as a PoC (Proof-of-Concept). The experiments were carried out with the fabricated PoC which was characterized to assess the trapping performance and determine the controllable experimental variables to tune the acoustofluidic effects aptly, leading to the formation of stable clusters in SBAW nodal planes. On successful fabrication, the device showed repeatable trapping over a wide bandwidth of 300 kHz, and trap stiffnesses of up to 1.7 fN/μm determined experimentally based on the processing of particle agglomerate imaging. It was observed that the trap stiffness was adequate for levitation over several hours, and yet allowed for the formation of a 3D agglomerate of particles. It was also shown that a frequency-sweep actuation was successful in overcoming fabrication limitations and suppressing streaming, and a suitable working range of experimental parameters could be determined to achieve initial clustering in under 3 minutes. A futuristic outlook on the in-plane particle confinement methods to further improve the target performance, and considerations for biological experiments as the immediate next step have been presented as a concluding note in this thesis. This work thus paves the way for the integration of this technique into laboratory organoid culture formats. This positively complements the objective of cutting down the agglomeration time for initial organoid clustering, for an anticipated positive impact on the production throughput and developmental aspects of organoid cultures, with the in-house fabricated and characterized PoC presented in this work as an enabler.

In-vitro 3D organoid cultures constitute an essential component of modern-day biological research, with key application areas in drug and therapy testing, and Organ-on-a-Chip models. The increasing demand for reliable models comes with the challenge of boosting the throughput of production of these cultures. The currently researched 3D culture methods pose the challenge of not being contact-free, of limited versatility and throughput in organoid culturing, and often lack a stimulus to promote cell agglomeration. This work presents acoustofluidics as a stimulus-driven solution to promote the clustering of cells in a levitated, suspended environment. In order to establish this objective experimentally, an SBAW resonator was designed, fabricated, and tested as a PoC (Proof-of-Concept). The experiments were carried out with the fabricated PoC which was characterized to assess the trapping performance and determine the controllable experimental variables to tune the acoustofluidic effects aptly, leading to the formation of stable clusters in SBAW nodal planes. On successful fabrication, the device showed repeatable trapping over a wide bandwidth of 300 kHz, and trap stiffnesses of up to 1.7 fN/μm determined experimentally based on the processing of particle agglomerate imaging. It was observed that the trap stiffness was adequate for levitation over several hours, and yet allowed for the formation of a 3D agglomerate of particles. It was also shown that a frequency-sweep actuation was successful in overcoming fabrication limitations and suppressing streaming, and a suitable working range of experimental parameters could be determined to achieve initial clustering in under 3 minutes. A futuristic outlook on the in-plane particle confinement methods to further improve the target performance, and considerations for biological experiments as the immediate next step have been presented as a concluding note in this thesis. This work thus paves the way for the integration of this technique into laboratory organoid culture formats. This positively complements the objective of cutting down the agglomeration time for initial organoid clustering, for an anticipated positive impact on the production throughput and developmental aspects of organoid cultures, with the in-house fabricated and characterized PoC presented in this work as an enabler.

Mechanical forces are integral to the functionality and behavior of biological systems, from the cellular to the molecular level. Acoustic force spectroscopy (AFS), an emerging field, seeks to explore these intricate dynamics. Traditional methods in AFS, particularly those using bulk acoustic wave (BAW) devices, still lack sample visibility and have a low throughput. These limitations hinder the amount and type of data that can be gathered in the study of cellular and molecular mechanics. This research project addresses the challenge of enhancing visibility and throughput in AFS by developing a novel BAW device. Here we show the fabrication and implementation of a glass transversal resonator by femtosecond laser ablation and a simple glass-glass bonding technique using polyethylene film and cyanoacrylate glue, which enables two-dimensional particle trapping. Compared to existing BAW setups in AFS, our approach offers improved sample visibility and is capable of positioning particles at arbitrary locations between half- and full-wave mode equilibria by rapid mode-switching. It shows that the position can be predicted using a straightforward method to determine the stiffness of each mode. This advancement of acoustic force spectroscopy could pave the way for new approaches in biomedical research. For example, the ability to alter the force field in a predictable manner allows to form both force- and distance-clamps. More functional BAW devices could thereby not only enhance our understanding of cellular processes but also find broader application in fields like tissue engineering and medical diagnostics.

Surface acoustic wave (SAW) is widely used in biological research and sensor applications, acomprehensive understanding of SAW propagation and visualization on suspended 2D membranes is crucial for the manipulation of nanoparticles in biological research and sensitivity improvement of SAW sensors based on 2D material. The initial step of this project involved exciting a standing SAW field on the SAW device and attempting to use Digital Holography Microscopy (DHM) for imaging the standing SAW field on the substrate. This attempt was very challenging and unfortunately was not successful. We used an Atomic Force Microscope (AFM) as an alternative solution for SAW field imaging. Initially, AFM was used to image the SAW field on the SAW device substrate, and the impacts of input frequency and power on the amplitude of the SAW field were investigated. Subsequently, microcavities were fabricated on the SAW device to record images of the SAW field within the microcavities and their surrounding areas, aiming to investigate the impact of microcavities on the SAW field. Finally, the prepared graphene membrane was transferred to the microcavities, the SAW propagation was recorded on the suspended membrane and the influence of the suspended membrane on SAW propagation was analyzed.