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Cell mechanics are directly related to the biological functionality of a cell, and therefore have been extensively studied. Current understanding of the unique relationships associated with mechanical loading conditions and the biological outcomes of a cell is far from complete [1]. The main objective of this thesis work was the design of a device capable of determining mechanical properties including stiffness and Young’s modulus of a biological cell. The device was implemented using micro-electro mechanical systems technology (MEMS), and the cell testing was limited to yeast cells for the purpose of this research. The design consisted of a micro-gripper which performed controlled cell squeezing with a spring of known stiffness. Differential displacements were obtained allowing for the calculation of cell mechanical properties. The incorporation of spatially periodic structures on the moving components of the gripper enabled measurements with 10 nm precision based on discrete Fourier transformation and phase [2].

Die mechanischen Eigenschaften von Zellen charakterisieren und beeinflussen deren Zustand. Die vorliegende Arbeit zielt auf ein besseres Verständnis der biomechanischen Eigenschaften von Zellen ab. Der Fokus lag dabei auf der Biegesteifigkeit von Zellmembranen und der Deformierbarkeit der Zellen. Es werden drei Studien vorgestellt in der diese Materialparameter untersucht wurden. Die erste Studie befasst sich mit der Temperaturabhängigkeit der mechanischen Eigenschaften. Hierbei wurden acht verschieden Zelllienien bei jeweils fünf Temperaturen rheologisch vermessen. Zur Messung wurde der sog. \"optical stretcher\" verwendet der gleichzeitig die Zellen deformieren und aufheizen kann. Die Versuche zeigen, dass eine Zeit-Temperatur superposition dabei nicht für alle Zelltypen funktioniert. In der zweiten Studie wurden die Membransteifigkeit von Gewebeproben von Brust- und Gebärmutterhalskrebspatienten untersucht. Als Kontrollsystem wurde gutartiges Gewebe aus dem Umfeld des Tumors verwendet. Es konnte gezeigt werden, dass die Zellmembranen von Tumorzellen weicher waren als von gesundem Vergleichsgewebe. Die Änderung der Membrankomposition wurde dabei als mögliche Ursache massenspektroskopisch Untersucht und verschieden Ursachen der weichen Membrane diskutiert. Für die dritte Studie wurde der chemische Wirkstoff Soraphen A eingesetzt um die Membransteifigkeit von zwei Zelllienien zu erhöhen. Dies zeigte eine Verringerung von Zellbeweglichkeit und Invasivität.

Cell-substrate interactions play a key role in the regulation of epithelial cell mechanics. Through a series of studies, we demonstrate how substrate interactions impact both the response to an oncogene and the cellular contractility and organization of a monolayer. We first examine the effects of oncogenic Ras in cells in adherent and suspended states. To accomplish this, we utilized atomic force microscopy and a microfluidic optical stretcher. We found that adherent cells stiffen and suspended cells soften with the expression of constitutively active Ras. The effect on adherent cells was reversed when contractility was inhibited with the ROCK inhibitor Y-27632, resulting in softer cells. These findings suggest that increased ROCK activity as a result of Ras has opposite effects on suspended and adhered cells. In a subsequent study, we examined the effects of a substrate on contracting and relaxing monolayers. We created a new methodology for measuring the mechanodynamics of epithelial monolayers by culturing cells at an air-liquid interface. These model monolayers were grown in the absence of any supporting structures in hanging drops. We found that the direction of strain in the unsupported monolayers was not correlated to nuclear alignment as observed when the monolayers were grown on soft deformable gels. It was also observed that both gel and glass substrates led to the promotion of long-range cell nuclei alignment not seen in the unsupported monolayers. To further characterize the morphology and mechanics of monolayers clusters observed in our experiments, we created a new computational model based on the vertex model. The energy function used in this model takes into account cell-cell and cell-substrate adhesion as well as anisotropic cellular mechanical properties. The results of these simulations suggest that the promotion of long-range alignment on solid substrates were due to cells having anisotropic elastic moduli with global alignment. They also suggest that the alignment observed in monolayers grown on air water interfaces is due to cells having low substrate adhesion and isotropic moduli. Our findings establish the importance of studying epithelial cell mechanics in different states of attachment.

A significant body of literature has reported strategies and techniques to assess the mechanical properties of biological samples such as proteins, cellular and tissue systems. Atomic force microscopy has been used to detect elasticity changes of cancer cells. However, only a few studies have provided a detailed and complete protocol of the experimental procedures and data analysis methods for non-adherent blood cancer cells. In this work, the elasticity of NB4 cells derived from acute promyelocytic leukemia (APL) was probed by AFM indentation measurements to investigate the effects of the disease on cellular biomechanics. Understanding how leukemia influences the nanomechanical properties of cells is expected to provide a better understanding of the cellular mechanisms associated to cancer, and promises to become a valuable new tool for cancer detection and staging. In this context, the quantification of the mechanical properties of APL cells requires a systematic and optimized approach for data collection and analysis, in order to generate reproducible and comparative data. This Thesis elucidates the automated data analysis process that integrates programming, force curve collection and analysis optimization to assess variations of cell elasticity in response to processing criteria. A processing algorithm was developed by using the IGOR Pro software to automatically analyze large numbers of AFM data sets in an efficient and accurate manner. In fact, since the analysis involves multiple steps that must be repeated for many individual cells, an automated and un-biased processing approach is essential to precisely determine cell elasticity. Different fitting models for extracting the Young’s modulus have been systematically applied to validate the process, and the best fitting criteria, such as the contact point location and indentation length, have been determined in order to obtain consistent results. The designed automated processing code described in this Thesis was used to correlate alterations in cellular biomechanics of cancer cells as they undergo drug treatments. In order to fully assess drug effects on NB4 cells, viability assays were first performed using Trypan Blue staining for primary insights before initiating thorough microplate fluorescence intensity readings using a LIVE/DEAD viability kit involving ethidium and calcein AM labelling components. From 0 to 24 h after treatment using 30 µM arsenic trioxide, relative live cell populations increased until 36 h. From 0 to 12 h post-treatment, relative populations of dead cells increased until 24 h post-treatment. Furthermore, a drastic drop in dead cell count has been observed between 12 and 24 h. Additionally, arsenic trioxide drug induced alterations in elasticity of NB4 cells can be correlated to the cell viability tests. With respect to cell mechanics, trapping of the non-adherent NB4 cells within fabricated SU8-10 microwell arrays, allowed consistent AFM indentation measurements up to 48 h after treatment. Results revealed an increase in cell elasticity up to 12 h post-treatment and a drastic decrease between 12 and 24 h. Furthermore, arsenic trioxide drug induced alterations in elasticity of NB4 cells can be correlated to the cell viability tests. In addition to these indentation and viability testing approaches, morphological appearances were monitored, in order to track the apoptosis process of the affected cells. Relationships found between viability and elasticity assays in conjunction with morphology alterations revealed distinguish stages of apoptosis throughout treatment. 24 h after initial treatment, most cells were observed to have burst or displayed obvious blebbing. These relations between different measurement methods may reveal a potential drug screening approach, for understanding specific physical and biological of drug effects on the cancer cells.

It has been well established that the fundamental behaviors of mammalian cells are influenced by the physical cues that they experience from their surrounding environment. With respect to cells in our bodies, mechanically-driven morphological and phenotypic changes to our cells have been linked to responses critical to both normal development and disease progression, including lung, heart, muscle and bone disorders, and cancer. Although significant advancements to our understanding of cell behavior have been made using 2D cell culture methods, questions regarding how physical stretch guides cell behavior in more complex 3D biological systems remain unanswered. To address these questions, we used microfabrication techniques to develop vacuum-actuated stretchers for high throughput stretching and dynamic mechanical screening of 3D microtissue cultures. This thesis contains five research chapters that have utilized these devices to advance our understanding of how cells feel stretch and how it influences their behavior in a 3D matrix. In the first research chapter (chapter 2), we characterized how stretch is transferred from the tissue-level to the single-cell level and we investigated the cytoskeletal reinforcement response to long-term mechanical conditioning. In the second research chapter (chapter 3), we examined the effects of an acute dynamic stretch and found that 3D cultures soften through actin depolymerization to homeostatically maintain a mean tension. This softening response to stretch may lengthen tissues in our body, and thus may be an important mechanism by which airway resistance and arterial blood pressure are controlled. In the third and forth research chapters (chapter 4-5), we investigated the time dependencies of microtissues cultures and we found that their behavior differed from our knowledge of the rheological behavior of cells in 2D culture. Microtissues instead followed a stretched exponential model that seemed to be set by a dynamic equilibrium between cytoskeletal assembly and disassembly rates. The difference in the behavior from cells in 2D may reflect the profound changes to the structure and distribution of the cytoskeleton that occur when cells are grown on flat surfaces vs. within a 3D environment. In the fifth and final research chapter (chapter 6), we examined how mechanical forces may contribute to the progression of tissue fibrosis through activating latent TGF-β1. Our results suggest that mechanical stretch contributes to a feed forward loop that preserves a myofibroblastic phenotype. Together these investigations further our understanding of how cells respond to mechanical stimuli within 3D environments, and thus, mark a significant contribution to the fields of mechanobiology and cell mechanics.

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Improving treatment and prevention of pelvic organ prolapse, a disorder affecting up to half of parous women, requires thorough mechanical analysis of the vagina and other endopelvic structures at the cellular level. In this study, we tested single vaginal smooth muscle cells (SMCs) to quantify their elastic moduli. Cells were enzymatically isolated from vaginal walls of freshly sacrificed, virgin Long Evans rats and cultured using well-established methods. A custom-built experimental setup was used to perform tensile tests. Micropipettes were fabricated to serve as cantilever-type load cells, which were coated in cellular adhesive. Two pipettes applied tension to SMCs until adhesion between the cell and a pipette failed. During mechanical testing, images of SMCs were collected and translated into strain and stress. Specifically, force/stress data were calculated using Euler-Bernoulli Beam Theory and by making simplifying geometric assumptions. The average initial and total elastic moduli (mean ± SEM) for single vaginal SMCs were 6.06 ± 0.26 kPa and 5.4 ± 0.24 kPa, respectively, which is within the range reported for other types of SMCs, mainly airway and vascular, of various species. This protocol can and will be applied to further investigate mechanics of single cells from the pelvic region with independent variables such as parity, age, body mass index, and various stages of POP. Results of these experiments will provide critical information for improving current treatments like drug therapies, surgical procedures, medical grafts and implants, and preventative practices like stretching and exercise techniques. / Master of Science

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