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This thesis aims to investigate the manipulation of cells, especially cancer cells, via acoustofluidic techniques at high ultrasound frequencies. This PhD project's motivation and ultimate goal are to separate circulating tumour cells (CTCs) from normal blood cells to achieve CTCs detection via acoustofluidic techniques. At the same time, the acoustofluidics-based manipulation of other types of cells and microparticles have also been investigated. \ud The presence of rare cancer cells in cancer patient blood, called CTCs, has been increasingly researched as an essential biomarker for cancer diagnosis and cancer treatment monitoring. Separation and enrichment of CTCs from cancer patients’ blood samples via liquid biopsy methods have shown excellent compatibility compared with the conventional screening and invasive tissue biopsy methods. As a novel, bio�compatible and label-free technique, acoustofluidics has the potential to become an effective tool to sort CTCs from liquid samples or manipulate other types of cells via the cells physical properties: size, density, and compressibility.\ud In this thesis, acoustofluidic platforms based on standing surface acoustic waves (SSAW) are demonstrated, including the Interdigital transducers (IDTs) design, cleanroom (CR) fabrication, and integration with microfluidics, electronics and mechanics systems. The simulation has been conducted via Governing equations (Continuity and Navier-Stokes equation) and Finite Element Method (FEM) model to understand the working principle and compare it with the microparticles manipulation experiment on the parallel and tilted-angle IDT SSAW devices. Moreover, a conventional tilted-angle (CTA) IDTs acoustofluidic device has been applied to wash the electroporated cells from the original medium, and a higher electroporation efficiency and cell viability are achieved. By optimising the IDTs patterning, a filled tilted-angle (FTA) IDTs design with less electrical input power but higher acoustic energy generated compared with CTA IDTs is demonstrated that achieves around 90% \ud deflection efficiency of Hela cells with the input power of 4.5 W. In addition, to overcome the challenges of frangibility and overheating due to the conventional SSAW substrates, a novel Gallium Nitride (GaN) compound semiconductor film based acoustic tweezer is demonstrated. Cancer cell patterning via the GaN platform has \ud been successfully achieved with excellent thermal stability with high input power. SSAW-based acoustofluidic cell manipulation in this thesis extends understanding of acoustofluidics techniques via the novel IDT design and SSAW generation substrate and will enable further development in high precision cell manipulation and biosensors applications

Human embryonic stem cells (hESCs) and their derived products offer great promise for targeted therapies and drug screening, however, the hESC differentiation process of mature neurons is a lengthy process. To accelerate the neuron production, an acoustic stimulator producing surface acoustic waves (SAWs) is proposed and realized by clamping a flexible printed circuit board (PCB) directly onto a piezoelectric substrate. Neural differentiation of the hESCs is greatly accelerated after application of the acoustic stimulations. Acceleration mechanisms for neural differentiation have been explored by bulk RNA sequencing, quantitative polymerase chain reaction (qPCR) and immunostaining. The RNA sequencing results show changes of extracellular matrix-related and physiological activity-related gene expression in the low or medium SAW dose group and the high SAW dose group, respectively. The neural progenitor cell markers, including Pax6, Sox1, Sox2, Sox10 and Nkx2-1, are less expressed in the SAW dose groups compared with the control group by the qPCR. Other genes including Alk, Cenpf, Pcdh17, and Actn3 are also found to be regulated by the acoustic stimulation. Moreover, the immunostaining confirmed that more mature neuron marker Tuj1-positive cells, while less stem cell marker Sox2-positive cells, are presented in the SAW dose groups. These results indicate that the SAW stimulation accelerated neural differentiation process. The acoustic stimulator fabricated by using the PCB is a promising tool in regulation of stem cell differentiation process applied in cell therapy. STATEMENT OF SIGNIFICANCE: Human embryonic stem cells (hESC) are used for investigating the complex mechanisms involved in the development of specialized biological cells and organs. Different types of hESCs derived cell products can be used for cell therapy procedures aiming to regenerate functional tissues in patients who suffer from various degenerative diseases. Accelerating the hESCs' differentiation process can considerably benefit the clinical utilization of these cells. This study develops a highly effective acoustic stimulator working at ∼20 MHz to investigate what roles do acousto-mechanical stimuli play in the differentiation of hESCs. Our results show that acoustic dose alters the extracellular matrix and physiological activity-related gene expression, which indicates that the acoustic stimulation is an important tool for regulating the stem cells' differentiation processes in cell therapy.