This thesis presents a combined numerical and experimental study on the response of kidney cells to shock waves. The motivation was to develop a mechanistic model of cell deformation in order to improve the clinical use of shock waves, by either enhancing their therapeutic action against target cells or minimising their impact on healthy cells. An ultra-high speed camera was used to visualise individual cells, embedded in tissue-mimicking gel, in order to measure their deformation when subject to a shock wave from a clinical shock wave source. Advanced image processing was employed to extract the contour of the cell from the images. The evolution of the observed cell contour revealed a relatively small deformation during the compressional phase and a much larger deformation during the tensile phases of a shock wave. The experimental observations were captured by a numerical model which describes the volumetric cell response with a bilinear Equation of State and the deviatoric cell response with a viscoelastic framework. Experiments using human kidney cancer cells (CAKI-2) and noncancerous kidney cells (HRE and HK-2) were compared to the model in order to determine their mechanical properties. The differences between cancerous and noncancerous cells were exploited to demonstrate a design process by which shock waves may be able to improve the specificity on targeted cancer cells while having minimal effect on normal cells. The cell response to shock waves was studied in a more biophysically realistic environment to include influence of cell size, shape and orientation, and the presence of neighbouring cells. The most significant difference was predicted when cells were in a cluster in which case the presence of neighbouring cells resulted in a four-fold increase on the von Mises stress and the membrane strain. Finally the numerical model was extended to capture the effect of cell damage using one of two paradigms. In the first paradigm the model captured microdamage during one shock wave but then assumed that the cell recovered by the time the next shock wave arrived. The second model allowed microdamage to accumulate with increasing number of shock waves. These models may be able to explain the strong effect that shock wave loading rate has on tissue damage. In conclusion a validated numerical model has been developed which provides a mechanistic understanding of how cells respond to shock waves. The model has application in suggesting improved strategies for current uses of shock waves, e.g., lithotripsy, as well as opening up new indications such as cancer treatment.

Therapeutics targeting the PI3K (phosphatidylinositol 3-kinase) and the Ras/MAPK (mitogen-activated protein kinases) pathways have potential as non-toxic treatments for triple-negative breast cancer due to their frequent over-activation in several forms of cancer. Interestingly, the PI3K and Ras/MAPK pathways have been shown to incite cancer dormancy behavior individually and tumorigenic behavior in unison when induced in healthy breast epithelial cells (MCF-10A) in vivo. Tumorigenesis and metastasis are heavily reliant on the specific mechanical and adhesive properties of cells, including decreased stiffness, increased mechanosensitivity, and decreased adhesion. However, the describe cellular behaviors are poorly understood for dormant cancer phenotypes. Understanding the mechanical and adhesive behaviors of MCF-10A cells as a function of PI3K and/or Ras/MAPK pathway over-activation further explores the cross-talk enabling unique dormant and tumorigenic characteristics. Cellular viscoelasticity and adhesion were measured for MCF-10A cells with PTEN (phosphatase and tensin homolog) knockout and activated KRAS (Kristen rat sarcoma viral oncogene homolog) overexpression to activate the PI3K and Ras/MAPK pathways respectively with atomic force microscopy. PTEN knockout alone has no observable influence on cell adhesion but resulted in softer cells with less organized cytoskeleton. Activated KRAS overexpression increased cell stiffness and cell adhesion regardless of PTEN expression level. Moreover, the overexpression of activated KRAS enhanced the sensitivity of cells to the substrate stiffness. The findings suggest that the cancer-associated pathways PI3K and Ras/MAPK regulate cell adhesion and mechanics to promote tumor formation and metastasis. More importantly, the results that signify mutations of different molecular pathways associated with cancer dormancy regulate cell mechanics differently suggests that cell stiffness is a biomarker that detects and differentiates different types of dormant cancers.

This thesis deals with the differential response of healthy and cancerous cells to surface topography at the nanoscale and the microscale. Using a statistical method we developed we studied the interactions of cells with grooves of nanoscale depth. We demonstrate that healthy cells have a greater ability to align with deeper grooves, whereas cancerous cells are more sensitive to shallow grooves. Analysis reveals that the nucleus follows the alignment of the cell body more closely in cancerous cells, and that the nucleus of cancerous cells is more sensitive to shallow grooves.On microscale pillars we demonstrate for the first time that osteosarcoma cells deform to adopt the surface topography and that the deformation extends to the interior of the cell and in particular to the nucleus. We show that healthy cells only deform during the initial stages of adhesion and that immortalized cells show intermediate deformation between the healthy and cancerous cells. When the spacing between the pillars is reduced, differences in the deformation of different cancerous cell lines are detected. Deformation was also found to be related to the malignancy in keratinocytes, and related to the expression of Cdx2 in adenocarcinoma. The mechanism of deformation is tentatively attributed to the cytoskeleton and attempts to identify the main actors of deformation were performed using confocal microscopy and cytoskeleton inhibitors. Live cell imaging experiments reveal that the deformed cells are very mobile on the surfaces, loss of deformation is necessary for mitosis to occur and deformation after mitosis is more rapid than initial deformation upon adhesion to surfaces.

No description available.

Most embryonic development and tissue self-assembly requires the integration of cell movements within multiple cell layers composed of different cell types, which are integrated with the signaling networks in these 3D environments. Although the role of cell mechanics in tissue self-assembly has been demonstrated, little is known about the mechanical responses of 3D multi-layer tissues to chemical cues. To investigate the collective movements within multilayered tissues, I developed a novel microfluidic technique capable of removing desired height or width of tissue from a composite tissue. I call this technique "3D tissue-etching" because it is analogous to techniques used in the microelectromechanics (MEMS) field where complex 3D structures are built by successively removing material from a monolithic solid through subtractive manufacturing. I used a custom-designed microfluidic control system to deliver a range of tissue etching reagents (detergents, chelators, proteases, etc.) to specific regions of multilayered tissues microsurgically isolated from embryos of the African Clawtoed frog, Xenopus laevis. Xenopus embryos and explanted tissues have long been used to elucidate signaling and other cellular processes during development and here provide an ideal model 3D tissue etching. Long exposure to a narrow etchant stream cuts completely through cell-cell layers to expose the substrate. By reducing the exposure time a single layer may be removed. By controlling the width of the etchant and the exposure time a broader swath of the surface layer may be removed. For more refined etching, after removal of a broad swath the resistance circuits can be switched and a second narrow stream can remove only a single narrow band within the swath exposed cells. I developed tissue-etching techniques that allow me to shape complex multi-layered embryonic tissues. The ability to control 3D stimulation and the form of multicellular tissues will provide extend the tools of tissue engineering to synthesize highly complex 3D integrated multicellular biosystems. Integration of tissue etching in my custom microfluidic system provides a "test-bed" where a range of hypotheses concerning the control and regulation of development and cell differentiation can be implemented and tested.

Die Plasmamembranspannung von eukaryotischen Zellen soll maßgeblich zur Regulation von zellulären Prozessen wie der Zellmigration, Mitose, Endo- und Exozytose, Membranreparatur, Osmoregulierung und Zellspreiten beitragen, welche zu einer Veränderung der Membranfläche und ihrer Deformation führt. In dieser Arbeit wurde die epitheliale Zelllinie MDCK II (Madin-Darby Canine Kidney) benutzt, um spannungsgesteuerte Oberflächenregulierung zu untersuchen. Indentationsexperimente kombiniert mit dem Herausziehen von Membrannanoröhren wurden mit Hilfe des Rasterkraftmikroskops (Atomic Force Microskope, AFM) durchgeführt, um lokale Variationen in der Membranspannung und überschüssiger Membranfläche als Funktion von äußeren Reizen abzuschätzen. Die verwendeten externen Stimuli beinhalten eine Veränderung der Funktionalität des Actomyosin-Cortexes durch die Wirkung von Blebbistatin und Cytochalasin D, sowie die Manipulation der Zytoskelett-Membran Adhäsionspunkte durch Einzel-Mikroinjektion. Die Injektion von Neomycin verhindert die Anbindung von ERM-Proteinen an das Lipid Phosphatidylinositol-(4,5)-bisphosphat (PIP2) und bewirkt somit die Abkopplung des Zytoskeletts von der Plasmamembran. Als Gegenexperiment diente die Injektion des Lipids PIP2 selbst, welches zur Erhöhung der Anzahl der Zytoskelett-Membran Adhäsionspunkte führte. Weiterhin wurden die als Membranreservoire dienenden Mikrovilli durch den Entzug von Cholesterol entfernt. Auswirkung auf das Vorhandensein von Membranreservoiren hat ebenfalls die Veränderung des osmotischen Drucks innerhalb der Zellen. Zusätzlich wurden die elastischen Eigenschaften von apikalen Zellmembran-Fragmenten von konfluenten MDCK II Zellen untersucht, welche Aufschluss über die intrinsischen Membraneigenschaften ohne den Einfluss des Zytosols und Zytoskeletts geben konnten. Abschließend wurde die Mechanik von adhärierenden und spreitenden Zellen untersucht. Zusammenfassend kann gesagt werden, dass die Plasmamembran, bestehend aus einer Phospholipiddoppelschicht, lateral schwer ausdehnbar ist aufgrund ihrer flüssig-kristallinen Natur. Durch das Vorhandensein von dynamischen Membranreservoiren wie Mikrovilli, die schnell auf Veränderungen der Membranspannung durch Membranhomöostase reagieren, werden zellulare Prozesse wie die Zellmotilität oder die Anpassung an osmotischen Stress ermöglicht. In der vorliegenden Arbeit gelang es gleichzeitig, die Membranspannung und die Verfügbarkeit von Membranfläche von adhärenten konfluenten als auch von adhärierenden und spreiten Zellen zu messen. Die durchgeführten Experimente ergaben ein detailliertes Bild wie sich die zelluläre Oberflächenregulierung in der Membranmechanik widerspiegelt.

The cellular mechanical environment, including the elasticity of the extracellular matrix, profoundly affects cellular mechanical and biological responses. This responsiveness depends on and may influence the inherent mechanical properties of the cell and the nucleus. In this thesis, the local and global elastic moduli of valve interstitial cells (VICs) cultured on substrates of varying stiffness were characterized using atomic force microscopy (AFM). A novel AFM technique used to directly determine nuclear elastic moduli in situ was also tested and preliminary results for VIC nuclear elasticity and isolated VIC nuclei elasticity were presented. This study confirmed that both local and global elasticity of VICs were sensitive to substrate compliance, and demonstrated that the nucleus was consistently two to four times stiffer than the cytoplasm and that isolated VIC nuclei were significantly softer than the intact nuclei in situ. It also provides practical guidelines for efficient AFM-based measurement of cell mechanical properties.

The cellular mechanical environment, including the elasticity of the extracellular matrix, profoundly affects cellular mechanical and biological responses. This responsiveness depends on and may influence the inherent mechanical properties of the cell and the nucleus. In this thesis, the local and global elastic moduli of valve interstitial cells (VICs) cultured on substrates of varying stiffness were characterized using atomic force microscopy (AFM). A novel AFM technique used to directly determine nuclear elastic moduli in situ was also tested and preliminary results for VIC nuclear elasticity and isolated VIC nuclei elasticity were presented. This study confirmed that both local and global elasticity of VICs were sensitive to substrate compliance, and demonstrated that the nucleus was consistently two to four times stiffer than the cytoplasm and that isolated VIC nuclei were significantly softer than the intact nuclei in situ. It also provides practical guidelines for efficient AFM-based measurement of cell mechanical properties.

No description available.

Diese Arbeit beinhaltet Ergebnisse der ersten klinischen Studie zur Charakterisierung der mechanischen Eigenschaften von Zellen in einem Tumor mit der dafür notwendigen Probengröße. Dies ermöglichte die Erstellung eines umfassenden Bildes von Subpopulationen innerhalb eines Tumors mit großem diagnostischem Potential. Die Änderung der Einzelzellmechanik von Tumorzellen wird durch Veränderung des Zytoskeletts, einem komplexes Polymernetzwerk in Zellen, hervorgerufen. Mit Hilfe von Zellgiften wurde das Zytoskelett gezielt manipuliert, um den Einfluss einzelner Faktoren auf die Biomechanik zu bestimmen. Aus Gewebeproben von Brustkrebspatienten wurden Zellen mit Hilfe enzymatischer Aufspaltung des extrazellulären Kollagennetzwerkes isoliert. Als Kontrollsystem wurden Primärzellen aus Brustreduktionsgewebe und aus Fibroadenomen, gutartigen Gewebeneubildungen der Brustdrüse, verwendet. Unter Einsatz des Optischen Stretchers, einer Zweistrahl-Laserfalle, wurden suspendierte Zellen für zwei Sekunden einer konstanten Zugspannung ausgesetzt und das Deformations- wie auch das anschließende Relaxationsverhalten beobachtet. Dabei ergaben sich wesentliche Unterschiede zwischen Tumor- und Kontrollproben. Neben Zellen mit ähnlichen Steifigkeiten, enthielten Tumorproben Subpopulationen sehr weicher Zellen, wie sie in Normalgewebe nicht zu finden sind. Desweiteren war das Relaxationsverhalten der Tumorzellen stärker elastisch dominiert. Einzelne Zellen kontrahierten sogar aktiv gegen die Zugspannung. Versuche, das Zytoskelett mittels Zellgiften künstlich in einem Zustand zu bringen, der in Krebszellen beobachtet wurde, ergaben zwar ebenfalls die Zunahme weicherer Zellen, jedoch war das Relaxationsverhalten eher viskos dominiert. Fluoreszenzaufnahmen des Aktin-Zytoskeletts sowie der fokalen Adhäsionen, die das Aktin-Netzwerk der Zelle mit dem Substrat verankern, zeigten Veränderungen bei Krebszellen im Vergleich zu Kontrollen. Darüber hinaus wurden Einflussfaktoren auf die Zellmechanik untersucht. Neben Kulturbedingungen, beeinflussen auch Alter und Medikation das biomechanische Verhalten. Die Steifigkeit der Krebszellen scheint vom Ursprungsgewebe beeinflusst zu werden, sodass Zellen verschiedener Krebsarten Steifigkeiten in unterschiedlichen Regimes zeigen. Die Ergebnisse dieser Arbeit liefern wichtige Informationen für unser Verständnis der Karzinogenese und bilden die Grundlage für eine neue diagnostische Methode zur Bestimmung der Tumoraggressivität. Eine gezielte Untersuchung der gefundenen Subpopulationen in einem Tumor könnte dabei helfen, neue Therapieansätze zu entwickeln und damit die hohen Rezidivraten aggressiver Tumore zu vermindern.