Acoustically focusing and measuring biological cells

Abstract

In the 1990s, thousand-year-old mummies were exhumed at an excavations site in the southern tip of Peru. The exhumed mummies included the mummified body of a woman in her mid thirties who presented an abnormal growth on the bone of her upper arm. Such bone tumors are classified as an orphan disease due to their low prevalence in the general public, have a peak incidence in children and adolescents, and have a high mortality rate. The high mortality rate stems, to a large degree, from cancer cells disseminating from the primary tumor and forming a secondary lesion in a process known as metastasis. Liquid biopsies, such as blood samples, therefore present a unique opportunity to isolate these disseminated tumor cells in order to gain patient specific diagnostic insight. An ideal method to isolate biological cells from liquid biopsies would not require labelling of the sample and would not diminish the cell viability. Acoustofluidics, a contactless and label-free method known to not decrease cell viability, exploits an acoustic field within a fluid cavity to manipulate objects in a fluid. Many variations of acoustofluidic devices have been developed, which include bulk acoustic wave (BAW) devices, where a standing pressure wave determines the position of objects within the fluid cavity of the BAW device. BAW devices have already been tested for the isolation of cancer cells in research settings. Although these preliminary studies demonstrated that BAW devices can be used to isolate cancer cells, there are limiting factors for BAW devices to be employed outside of research settings. This thesis focuses on how BAW devices can be improved to successfully isolate cancer cells from a liquid biopsy and how to measure the dynamic material properties of biological cells. An actuating element, usually a piezoelectric transducer manually attached to the BAW device, is excited at a frequency at which a standing pressure wave can be established in the fluid cavity. The continuous focusing and subsequently isolation of cancer cells from fluids therefore prerequisites that the optimal excitation frequency to establish a standing pressure wave can be found and dynamically altered. Many approaches are found in literature how to achieve a stable optimal excitation frequency, such as improving the BAW device design and fabrication. These approaches however only optimize the theoretical focusing efficiency. Another approach is to take the real time video feed of the focusing of the objects in the fluid as the control parameter of a feedback control loop (FCL). This thesis details the implementation of a FCL which can dynamically alter the excitation frequency in order to minimize the object distribution, quantified by the light intensity distribution, within the fluid cavity without increasing the cost or complexity of the system. The suggested FCL is not only straightforward and autonomous, it furthermore outperforms a skilled human operator. The FCL performed tasks otherwise difficult, such as focusing 600 nm diameter polystyrene particles in flow, whereas the optical system was the limiting factor to determine how small the objects could be and still be focused. Future work building upon this FCL could include looking into changing the input variable, e.g. transitioning from a video feed to a light sheet, which could reduce the space requirements and could help to bring BAW devices closer to bedside applications. Furthermore, the FCL could be altered for the use in device multiplexing, cell patterning or organoid formation, depending on the input parameter. Isolating biological cells is however only a first step. An ever growing research field focuses on measuring the material properties of biological cells. Knowing the material properties can lead to an increased diagnostic insight and is crucial information when designing acoustofluidic systems which rely on the relative compressibility and density differences between the object in the fluid and the fluid, which is characterized by the acoustic contrast factor (ACF). The hypothesis is explored if known static material properties of a bone cancer cell line with low and high metastatic potential can be used as a predictor for the dynamic material properties. The cancer cells were placed in an acoustic field and their movement is compared to reference objects in the fluid in order to calculate the dynamic material properties of the cancer cells. The study did not show a difference in the dynamic material properties between the two metastatic potentials. This is highly relevant as the cancer cells used in this study decrease in size as they become more malignant. The higher metastatic potential cancer cells will therefore be harder to manipulate, due to their smaller size but similar ACF, which is a crucial takeaway. Furthermore, this study includes a more in depth analysis, by altering the stiffness of the parental cell line and implementing a numerical model, which demonstrates the influence of various material properties, such as the stiffness, compressibility and density. The insights gained during this thesis highlight the gap in the understanding of the coupling between static and dynamic material properties, which needs to be addressed. The woman with the bone tumor would not have been cured by acoustofluidic devices. But the research presented here could have helped in detecting her tumor and led to insights about her disease progression.