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Development of isolated island micropatterns for investigating cellular biomechanicsBunde, Katie A. 23 May 2024 (has links)
The ability of cells to probe their mechanical environment and react to external stimuli is critical for maintenance of their normal structure and function. Through connections to the extracellular matrix, cells can sense mechanical cues such as substrate rigidity and stretch, and through force transmission across their contractile cytoskeleton can react accordingly to those signals by applying contractile traction forces to their surrounding environment. Healthy cells can react to these mechanical cues to maintain their cytoskeletal prestress (tension) at a set or homeostatic level over time, a phenomenon known as tensional homeostasis. Progression of certain diseases such as asthma, atherosclerosis, and cancer have been linked to a loss of tensional homeostasis. As such, tools for quantifying the traction forces that adhered cells apply to their substrate are crucial for gaining a better understanding of not just how healthy cells interact mechanically with their environment, but also how changes to the extracellular matrix or mutations within the cell can impact their ability to maintain tensional homeostasis and therefore remain both functional and viable. Our group has previously developed a method of quantifying cellular traction forces using indirectly pattered, soft hydrogel substrates known as micropattern traction microscopy. This method was initially developed to create discrete grid micropatterns, which while useful for measuring cellular traction forces does not offer any ability for the user to control cell growth area shape or size. This technique was further improved on through the creation of a protocol for changing discrete grid patterns into isolated island micropatterns, but this two-step process was challenging and generated islands of inconsistent shape and size. Here, we propose a new method for generating isolated island micropatterns of essentially any desired shape and size in a single step, as well as a corresponding image analysis algorithm for calculating cellular traction forces from these island micropatterns.
Additionally, this dissertation also includes an investigation into the impact of distinct Epithelial-cadherin mutations on the ability of gastric adenocarcinoma (AGS) cells to achieve tensional homeostasis. Disruption of tensional homeostasis in the epithelium is a hallmark of certain cancers, and mutations in E-cadherin proteins have been identified in malignant epithelial cells. Here, through analysis of AGS cell traction force data collected previously by Dr. Han Xu during her dissertation work, we have found that two distinct mutations in the intracellular domain of E-cadherins have an impact on the ability of AGS cells to achieve tensional homeostasis.
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An improved approach for cell traction force microscopy using a continuous hydrogelShojaeizadeh, Mina 06 June 2013 (has links)
"In this thesis, a cell traction force microscopy method is developed for measuring traction forces of connective tissue cells. This method includes an improved methodology in traction force microscopy of live cells cultured on an elastic substrate. Tissue cells, such as skin and muscle cells respond to the mechanical stimuli of their microenvironment by adhering to their substrate and exerting forces on the proteins of the extracellular matrix (ECM). These forces are called cell traction forces. Fibroblasts are grown on polyacrylamide (PA) gels embedded with fluorescent beads and coated with different types of ECM ligands. Traction forces of NIH 3T3 fibroblasts are calculated from the measured deformations of PA gels by using a 3-D finite element method. The advantages of this method compared to the traditional methods of cell traction force microscopy (CTFM) are that this method takes into account the finite thickness of the substrate by applying a 3-D FEM analysis to reduce the errors of using an infinite half space approximation for a substrate with a finite thickness and that it uses a novel method for embedding the substrate with fluorescent markers that decreases the measurement uncertainties. In our approach fluorescent beads were embedded on the top of substrate instead of getting mixed with the gel. This decreases the effect of out-of-focus fluorescent beads on the measured deformation fields which enhances the accuracy of cell traction force measurements."
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Forces mécaniques au sein de l'endothélium / Mechanical forces within endotheliumMoussus, Michel 05 February 2014 (has links)
Les dysfonctionnements vasculaires ou les blessures induites par l'âge, le tabac, les traumatismes ou une hyperlipidémie font partie de la myriade de facteurs de risques qui contribuent à la pathogénèse de nombreuses maladies cardiovasculaires. Un objectif important de la biologie vasculaire est de comprendre les processus cellulaires qui favorisent ou protègent contre ces maladies vasculaires. Cette pathogénèse est étroitement associée avec le dysfonctionnement de la paroi interne des vaisseaux sanguins. Cette paroi est constituée par une monocouche de cellules endothéliales qui forment l'endothélium vasculaire. La réparation de l'endothélium implique le remodelage des adhésions focales (AF) et des jonctions adhérentes (JA). Des modifications dans la composition protéique de ces structures adhésives génèrent des forces qui sont à la base du remodelage et de la réparation de l'endothélium. Dans la littérature, les forces cellulaires sont étudiées sur des cellules isolées, des doublets de cellules ou des ilots de cellules en croissance mais les forces mécaniques qui s'exercent au sein d'un tissu doivent encore être caractérisées. Dans cette thèse, nous utilisons la Microscopie à Traction de Force (TFM) sur des substrats en polyacrylamide pour étudier l'équilibre mécanique entre les jonctions intercellulaires et les adhésions cellule/substrat. Nous analysons dans quel mesure la TFM peut être utilisée pour étudier des monocouches cellulaires et présentons une nouvelle approche pour extraire les forces contractiles exercées par un tissu endothéliale. Finalement, nous utilisons cette méthode pour caractériser les forces transmises par les cellules à leur substrat et les forces contractiles pour une monocouche endothéliale. Cette méthode fournit un outil intéressant pour étudier la contribution de certaines protéines des jonctions adhérentes sur les forces transmises au sein de l'endothélium. / Vascular dysfunction or injury induced by aging, smoking, inflammation, trauma, hyperlipidaemia are among a myriad of risk factors that contribute to the pathogenesis of many cardiovascular diseases. An important objective in vascular biology is to understand cellular processes that promote or protect against cardiovascular diseases. This pathogenesis is very closely associated with dysfunction of the inner face of the vessel wall. The inner face of the vessel wall is lined by a monolayer of endothelial cells forming the vascular endothelium. Reparation of the endothelium involves remodelling of focal adhesionns (FA) and adherent junctions (AJ). Modifications in the protein composition of these adhesive structures generate forces at the basis of endothelium remodelling and reparation. In the literature, cellular forces are studied on single cells, epithelial cell doublets or cell aggregates in growth but mechanical forces inside tissues remains to be characterized. In this thesis, we use traction force microscopy (TFM) on polyacrylamide substrates to study the mechanical equilibrium between intercellular junctions and cell/substrate adhesion. We analyse to which extent TFM can be used for studying monolayers and present a novel approach to extract contractile forces exerted by an endothelial tissue. Finally, we use this methodology to characterize forces transmitted to the substrate and the contractile forces of endothelial monolayers. This method provides an interesting tool to study the contribution of some proteins of the adherent junctions to force transmission within the endothelium.
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Mechanical Regulation of Apoptosis and Calcification within Valvular Interstitial CellsCirka, Heather Ann 28 April 2016 (has links)
Calcific aortic valvular disease (CAVD) is the most common valvular pathology in the developed world. CAVD results in calcifications forming on the aortic valve leaflets, inhibiting proper closure and causing complications of stenosis and regurgitation. Although, the mechanisms behind the disease initiation are unknown, it is believed to be a cell-mediated phenomenon, and not the result of passive degradation of the valve as once believed due to the increased prevalence with age. Currently, there are no pharmaceutical options for the prevention or reversal of calcifications, the only treatment option is complete valve replacement, an imperfect solution. Hindering the development of potential therapeutics is that currently there are no adequate animal models which replicate the calcification and cell death seen in disease explanted valves. An in vitro model has been develop where valvular interstitial cells (VICs), the main cell type of the valve, are seeded at high density into tissue culture polystyrene dishes and cultured with TGF-β1. This results in VICs activating to the myofibroblast phenotype and forming cell aggregates. Due to currently unknown mechanisms, apoptosis occurs within the center of the aggregates and calcification ensues. Although simplistic, this model has been used to show that rate and frequency of aggregation is affected by cellular tension; conditions of high tension increase aggregation response, while conditions of low tension prevent aggregation and calcification from occurring. It is important to note; however, that despite its wide usage, the current model is limited as the aggregation and subsequent calcification are random occurrences and are not consistent across literature where same conditions for control samples are used. The motivation of the presented work is two-fold. First, high intracellular tension has been suggested as one of the mechanisms leading to disease in the valve. Despite the clear and important role of cell tension, VIC tension has never before been measured in a dynamic environment. The ways in which dynamic stimulation affects individual VIC tension is not known. In aim one, a method is developed to allow for long-term cyclic stretch of VICs with measurement of cell traction force. It was found that cyclic stretch decreased cell tension in cells with high prestress and increased cell tension for conditions of low prestress. Combined, these findings indicate a homeostatic cellular tension which is dependent upon the mechanical environment. In the second aim, a novel method for creating VIC aggregates is validated. Micro-contact printing, essentially “stampingâ€� of a protein in a defined pattern, is used to create circular aggregates on polyacrylamide gels. This method allows for the separation of the aggregation from the subsequent calcification, an improvement over the current in vitro model. The method is then used to explore the role of the distribution of tension in the initiation of disease
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Investigating Mechanotransduction and Mechanosensitivity in Mammalian CellsAl-Rekabi, Zeinab 02 December 2013 (has links)
Living organisms are made up of a multitude of individual cells that are surrounded by biomolecules and fluids. It is well known that cells are highly regulated by biochemical signals; however it is now becoming clear that cells are also influenced by the mechanical forces and mechanical properties of the local microenvironment. Extracellular forces causing cellular deformation can originate from many sources, such as fluid shear stresses arising from interstitial or blood flow, mechanical stretching during breathing or compression during muscle contraction. Cells are able to sense variations in the mechanical properties (elasticity) of their microenvironment by actively probing their surroundings by utilizing specialized proteins that are involved in sensing and transmitting mechanical information. The actin cytoskeleton and myosin-II motor proteins form a contractile (actomyosin) network inside the cell that is connected to the extracellular microenvironment through focal adhesion and integrin sites. The transmission of internal actomyosin strain to the microenvironment via focal adhesion sites generates mechanical traction forces. Importantly, cells generate traction forces in response to extracellular forces and also to actively probe the elasticity of the microenvironment. Many studies have demonstrated that extracellular forces can lead to rapid cytoskeletal remodeling, focal adhesion regulation, and intracellular signalling which can alter traction force dynamics. As well, cell migration, proliferation and stem cell fate are regulated by the ability of cells to sense the elasticity of their microenvironment through the generation of traction forces. In vitro studies have largely explored the influence of substrate elasticity and extracellular forces in isolation, however, in vivo cells are exposed to both mechanical cues simultaneously and their combined effect remains largely unexplored. Therefore, a series of experiments were performed in which cells were subjected to controlled extracellular forces as on substrates of increasing elasticity. The cellular response was quantified by measuring the resulting traction force magnitude dynamics. Two cell types were shown to increase their traction forces in response to extracellular forces only on substrates of specific elasticities. Therefore, cellular traction forces are regulated by an ability to sense and integrate at least two pieces of mechanical information - elasticity and deformation. Finally, this ability is shown to be dependent on the microtubule network and regulators of myosin-II activity.
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Investigating Mechanotransduction and Mechanosensitivity in Mammalian CellsAl-Rekabi, Zeinab January 2013 (has links)
Living organisms are made up of a multitude of individual cells that are surrounded by biomolecules and fluids. It is well known that cells are highly regulated by biochemical signals; however it is now becoming clear that cells are also influenced by the mechanical forces and mechanical properties of the local microenvironment. Extracellular forces causing cellular deformation can originate from many sources, such as fluid shear stresses arising from interstitial or blood flow, mechanical stretching during breathing or compression during muscle contraction. Cells are able to sense variations in the mechanical properties (elasticity) of their microenvironment by actively probing their surroundings by utilizing specialized proteins that are involved in sensing and transmitting mechanical information. The actin cytoskeleton and myosin-II motor proteins form a contractile (actomyosin) network inside the cell that is connected to the extracellular microenvironment through focal adhesion and integrin sites. The transmission of internal actomyosin strain to the microenvironment via focal adhesion sites generates mechanical traction forces. Importantly, cells generate traction forces in response to extracellular forces and also to actively probe the elasticity of the microenvironment. Many studies have demonstrated that extracellular forces can lead to rapid cytoskeletal remodeling, focal adhesion regulation, and intracellular signalling which can alter traction force dynamics. As well, cell migration, proliferation and stem cell fate are regulated by the ability of cells to sense the elasticity of their microenvironment through the generation of traction forces. In vitro studies have largely explored the influence of substrate elasticity and extracellular forces in isolation, however, in vivo cells are exposed to both mechanical cues simultaneously and their combined effect remains largely unexplored. Therefore, a series of experiments were performed in which cells were subjected to controlled extracellular forces as on substrates of increasing elasticity. The cellular response was quantified by measuring the resulting traction force magnitude dynamics. Two cell types were shown to increase their traction forces in response to extracellular forces only on substrates of specific elasticities. Therefore, cellular traction forces are regulated by an ability to sense and integrate at least two pieces of mechanical information - elasticity and deformation. Finally, this ability is shown to be dependent on the microtubule network and regulators of myosin-II activity.
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Design of Modified Traction Force Microscopy for Cell Response to De Novo ECMGnanasambandam, Bhargavee 07 September 2020 (has links)
No description available.
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ACCELERATED CELLULAR TRACTION CALCULATION BY PREDICTIONS USING DEEP LEARNINGIbn Shafi, Md. Kamal 01 December 2023 (has links) (PDF)
This study presents a novel approach for predicting future cellular traction in a time series. The proposed method leverages two distinct look-ahead Long Short-Term Memory (LSTM) models—one for cell boundary and the other for traction data—to achieve rapid and accurate predictions. These LSTM models are trained using real Fourier Transform Traction Cytometry (FTTC) output data, ensuring consistency and reliability in the underlying calculations. To account for variability among cells, each cell is trained separately, mitigating generalized errors. The predictive performance is demonstrated by accurately forecasting tractions for the next 30-time instances, with an error rate below 7%. Moreover, a strategy for real-time traction calculations is proposed, involving the capture of a bead reference image before cell placement in a controlled environment. By doing so, we eliminate the need for cell removal and enable real-time calculation of tractions. Combining these two ideas, our tool speeds up the traction calculations 1.6 times, leveraging from limiting TFM use. As a walk forward, prediction method is implemented by combining prediction values with real data for future prediction, it is indicative of more speedup. The predictive capabilities of this approach offer valuable insights, with potential applications in identifying cancerous cells based on their traction behavior over time.Additionally, we present an advanced cell boundary detection algorithm that autonomously identifies cell boundaries from obscure cell images, reducing human intervention and bias. This algorithm significantly streamlines data collection, enhancing the efficiency and accuracy of our methodology.
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Investigations of the spreading and closure mechanisms of phagocytosis in J774a.1 macrophagesKovari, Daniel T. 27 May 2016 (has links)
Phagocytosis is the process by which cells engulf foreign bodies. It is the hallmark behavior of white blood cells, being the process through which those cells ingest and degrade pathogens and debris. To date a large amount of research has focused on documenting the existence and role of biochemical components involved with phagocytosis. Scores of signaling molecules have been implicated in the complex signal cascade which drives the process. These molecules are small (typically no larger than 5 nanometers) and operate in a crowded, chemically “noisy,” environment, yet they coordinate the cell's activity over comparatively expansive distances (as large as 20 micrometers). How these molecular processes scale-up to coordinate the activities of the cell over such massive distances is largely unknown. Using a planar analog of phagocytosis termed “frustrated phagocytosis,” we experimentally demonstrate that phagocytosis occurs in three distinct phases: initial cell-antigen binding, symmetric spreading, and late-stage contraction. Initial binding and symmetric spreading appears to be both mechanically and chemically similar to the quasi-universal cellular behaviors of adhesion and migration. Adhesion and migration have received extensive attention from the biophysics community in recent years. Leveraging these similarities, we adapt the biomechanical frameworks used in models of migration to phagocytosis. We show that macroscopic properties such as a cell's effective viscosity and membrane cortical tension can be used to model cell behavior during phagocytosis. Our experiments reveal that late-stage contraction distinguishes frustrated phagocytosis from other spreading behaviors. This contraction is myosin dependent. Additionally we demonstrate, for the first time, that late-stage contraction corresponds with formation of a contractile F-actin belt. Based on the dynamic contraction model (DC) developed to explain actin structure during cell migration we propose a DC model of phagocytosis which posits that contractile belt formation is the result of a late-stage myosin activity coupled with F-actin.
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The Influence of Substrate Elasticity and Shear Rate on Human Blood Platelet Contraction / Time Resolved Data Acquisition, Microfluidic Designs and AlgorithmsHanke, Jana 20 April 2018 (has links)
No description available.
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