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Analysis of the Influence of Cellular Phase on Cell Traction Force MagnitudesFranklin, Jared Matthew 01 June 2015 (has links)
"Cell traction force is generated in the cytoskeleton by actomyosin activity and plays an important role in many cellular processes. In previous cell traction force experiments performed by our lab, unexpectedly large variations were measured. Because these experiments were utilizing a cell population of randomized phase, and there had been no documented investigation into whether cell phase affected cell traction force generation or propagation, it was hypothesized that there would be a significant difference in traction force between S phase and the other phases of interphase, as the physical and chemical changes happening within the nucleus at this time might elicit changes within the cytoskeleton. To test this hypothesis, we characterized the time-evolution of traction forces from a population of synchronized 3T3 fibroblasts. 3T3 fibroblasts were synchronized in G1-phase via serum starvation. The transition times between cellular phases during the first cell cycle after synchronization were identified by BrdU and Hoechst staining at different time points. After phase transition times were approximated, the traction forces of 9 cells were measured in 4-hour intervals for 24 hours. The differences between traction forces measured in G1, S, and G2 phases are not significant, demonstrating that cellular phase does not significantly affect traction force magnitude."
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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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Cell Traction Force Mapping in MG63 and HaCaTsSoon, Chin Fhong, Genedy, Mohamed A., Youseffi, Mansour, Denyer, Morgan C.T. January 2013 (has links)
No / The ability of a cell to adhere and transmit traction forces to a surface reveals the cytoskeleton integrity of a cell. Shear sensitive liquid crystals were discovered with new function in sensing cell traction force recently. This liquid crystal has been previously shown to be non-toxic, linear viscoelastic and sensitive to localized exerted forces. This paper reports the possibility of extending the application of the proposed liquid crystal based cell force sensor in sensing traction forces of osteoblast-like (MG-63) and human keratinocyte (HaCaT) cell lines exerted to the liquid crystal sensor. Incorporated with cell force measurement software, force distributions of both cell types were represented in force maps. For these lowly contractile cells, chondrocytes expressed regular forces (10 – 90 nN, N = 200) around the circular cell body whereas HaCaT projected forces (0 – 200 nN, N = 200) around the perimeter of poly-hedral shaped body. These forces are associated with the organisation of the focal adhesion expressions and stiffness of the LC substrate. From the results, liquid crystal based cell force sensor system is shown to be feasible in detecting forces of both MG63 and HaCaT.
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Tracking Traction Force Changes of Single Cells on the Liquid Crystal SurfaceSoon, Chin Fhong, Tee, K.S., Youseffi, Mansour, Denyer, Morgan C.T. 02 December 2014 (has links)
Yes / Cell migration is a key contributor to wound repair. This study presents findings indicating that the liquid crystal based cell traction force transducer (LCTFT) system can be used in conjunction with a bespoke cell traction force mapping (CTFM) software to monitor cell/surface traction forces from quiescent state in real time. In this study, time-lapse photo microscopy allowed cell induced deformations in liquid crystal coated substrates to be monitored and analyzed. The results indicated that the system could be used to monitor the generation of cell/surface forces in an initially quiescent cell, as it migrated over the culture substrate, via multiple points of contact between the cell and the surface. Future application of this system is the real-time assaying of the pharmacological effects of cytokines on the mechanics of cell migration.
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Development of a novel cell traction force transducer based on cholesteryl ester liquid crystals. Characterisation, quantification and evaluation of a cholesteryl ester liquid crystal based single cell force transducer system.Soon, Chin Fhong January 2011 (has links)
In biomechano-transducing, cellular generated tension can be measured by soft substrates based on polymers but these techniques are limited either by spatial resolution or ability to detect localised cell traction forces (CTF) due to their non-linear viscous behaviour under shear rates. A newly developed cell traction force transducer system based on cholesteryl ester lyotropic liquid crystals (LCTFT) was developed to sense localised traction forces of human keratinocyte cell lines (HaCaTs), in which the length of the deformation line induced represents the intensity of the CTF exerted. The physical properties of the cholesteryl ester based lyotropic liquid crystals (LLC) were characterised by using polarising microscopy, rheology, atomic force microscopy (AFM) based nano-indentation, spherical indentation, and micro-tensile tests. The interactions of LLC with cells were studied by using cell viability studies, cytochemical treatments, widefield surface plasmon resonance (WSPR) microscopy and various immuno-staining techniques. The results show that LLC is thermally stable (0 - 50 oC) and linearly viscoelastic below 10 % shear strain at shear rates of < 1 s-1. AFM nano and spherical indentations show a good agreement on the Young¿s modulus of both determined at ~110 kPa which is close to the elastic modulus of the epidermis. The Poisson¿s ratio of LLC was determined at ~0.58 by using micro tensile tests. The biophysical interaction studies indicated that LLC is biocompatible and allowed cell attachment. Cell relaxation technique by cytochalasin-B treatment suggested that the attachment and contraction of cells on LLC was due to the contractile activity of actin cytoskeletons that are mediated by focal adhesions. The staining experiments showed that cells consistently expressed the same suites of integrins (¿2, ¿3, ¿5 and ¿1) and ECM proteins (collagen type IV, laminin and fibronectin) on both glass and LLC coated substrates. Interfacial interaction of cells with LLC observed via the staining of actin and vinculin, and WSPR imaging suggest the association of marginal actin filaments and focal adhesions in attaching HaCaT cells to the LLC. Linear static analysis applied in the Finite Element model of focal adhesion-LC confirmed the compressive force patterns induced by cells. By applying cell relaxation techniques and Hooke¿s theorem, the force-deformation relationships of the LLC were derived and used for direct quantification of CTF in culture. The sensitivity of the LCTFT was implied by a wide range of CTF (10 - 140 nN) measured at high resolutions (~2 ¿m). Nonetheless, a custom-built cell traction force measurement and mapping software (CTFM) was developed to map CTF of single cells. Reliability of the LCTFT was evaluated by using a known pharmacological active cytokine, TGF-¿1, in inducing contraction of human keratinocytes. This study inferred internal consistency and repeatability of the LCTFT in sensing contraction responses of HaCaT cells in a concentration dependent manner of TGF-¿1. The overall LCTFT and CTFM software had shown good potential for use in the study of contraction and migration of keratinocytes. / Malaysia Ministry of Higher Education
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Development of a novel cell traction force transducer based on cholesteryl ester liquid crystals : characterisation, quantification and evaluation of a cholesteryl ester liquid crystal based single cell force transducer systemSoon, Chin Fhong January 2011 (has links)
In biomechano-transducing, cellular generated tension can be measured by soft substrates based on polymers but these techniques are limited either by spatial resolution or ability to detect localised cell traction forces (CTF) due to their non-linear viscous behaviour under shear rates. A newly developed cell traction force transducer system based on cholesteryl ester lyotropic liquid crystals (LCTFT) was developed to sense localised traction forces of human keratinocyte cell lines (HaCaTs), in which the length of the deformation line induced represents the intensity of the CTF exerted. The physical properties of the cholesteryl ester based lyotropic liquid crystals (LLC) were characterised by using polarising microscopy, rheology, atomic force microscopy (AFM) based nano-indentation, spherical indentation, and micro-tensile tests. The interactions of LLC with cells were studied by using cell viability studies, cytochemical treatments, widefield surface plasmon resonance (WSPR) microscopy and various immuno-staining techniques. The results show that LLC is thermally stable (0-50 °C) and linearly viscoelastic below 10% shear strain at shear rates of < 1 s⁻¹. AFM nano and spherical indentations show a good agreement on the Young's modulus of both determined at ~110 kPa which is close to the elastic modulus of the epidermis. The Poisson's ratio of LLC was determined at ~0.58 by using micro tensile tests. The biophysical interaction studies indicated that LLC is biocompatible and allowed cell attachment. Cell relaxation technique by cytochalasin-B treatment suggested that the attachment and contraction of cells on LLC was due to the contractile activity of actin cytoskeletons that are mediated by focal adhesions. The staining experiments showed that cells consistently expressed the same suites of integrins (α2, α3, α5 and β1) and ECM proteins (collagen type IV, laminin and fibronectin) on both glass and LLC coated substrates. Interfacial interaction of cells with LLC observed via the staining of actin and vinculin, and WSPR imaging suggest the association of marginal actin filaments and focal adhesions in attaching HaCaT cells to the LLC. Linear static analysis applied in the Finite Element model of focal adhesion-LC confirmed the compressive force patterns induced by cells. By applying cell relaxation techniques and Hooke's theorem, the force-deformation relationships of the LLC were derived and used for direct quantification of CTF in culture. The sensitivity of the LCTFT was implied by a wide range of CTF (10 - 140 nN) measured at high resolutions (~2 μm). Nonetheless, a custom-built cell traction force measurement and mapping software (CTFM) was developed to map CTF of single cells. Reliability of the LCTFT was evaluated by using a known pharmacological active cytokine, TGF-β1, in inducing contraction of human keratinocytes. This study inferred internal consistency and repeatability of the LCTFT in sensing contraction responses of HaCaT cells in a concentration dependent manner of TGF-β1. The overall LCTFT and CTFM software had shown good potential for use in the study of contraction and migration of keratinocytes.
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Struktur-Funktion-Wechselwirkungen in lateral eingeschränkten ZellenMüller, Andreas 04 November 2020 (has links)
Die Zellform ist wichtig für die Ausübung der Zellfunktion und spielt darüberhinaus eine essenzielle Rolle bei der Entwicklung eines Zellhaufens zu einem mehrzelligen Organismus. Dabei wird die Zellform neben biochemischen auch von biophysikalischen Prozessen beeinflusst: Zellkräfte sind ebenso beteiligt wie räumliche Einschränkung. Der Umfang der Wechselwirkung zwischen Umgebung, Zellform und Zellfunktion ist jedoch im Detail oft unverstanden. Ziel dieser Arbeit war daher, eine umfassende Charakterisierung von Zellen in räumlicher Einschränkung durchzuführen, um Aussagen zur Beeinflussung von Zellmorphologie und der Kraftentwicklung zu gewinnen.
In dieser Arbeit wurde die Reaktion humaner Primärzellen (HUVECs) auf laterale Einschränkung untersucht. Die Zellen wurden dafür sowohl auf Glas- als auch auf Hydrogel-Substraten kultiviert, die mittels Mikrokontaktdruck von Fibronektin mit Streifenmustern im Breitenbereich von 5μm bis 80μm strukturiert worden waren. Die Zellen wurden nach der Phase der initialen Adhäsion (> 1 h) hinsichtlich ihrer allgemeinen Morphologie, des Erscheinungsbildes ihres Aktinskeletts und ihres Zellzugkraftverhaltens quantitativ beschrieben. Zusätzlich erfolgten Lebendzellmessungen, um die Dynamik des Aktinskeletts und der Zellzugkräfte zu charakterisieren.
Die laterale Einschränkung führte zur strukturellen und funktionellen Adaption der Zellen. Da die Zelllänge nur geringfügig von der Streifenbreite abhing, kam es durch die seitliche Einschränkung zu einer Flächenabnahme bei gleichzeitiger Erhöhung des Zellseitenverhältnisses, wovon auch der Zellkern betroffen war. Die Ausrichtung der Aktinfasern korrelierte stark mit der Zellelongation und Zellen auf schmalen Streifen zeigten ein geringer vernetztes Aktinskelett. Messungen der Aktindynamik ergaben einen einwärts gerichteten Transport von Stressfasern. Weiterhin wurde eine Abnahme der Zugkräfte mit zunehmender Einschränkung gemessen, während gleichzeitig eine Polarisierung der Zugkräfte stattfand. Das beobachtete Verhalten der struktur- und funktionsbezogenen Zellparameter konnte gut durch die laterale Einschränkung erklärt werden, sodass die vorliegende Arbeit zu einem besseren Verständnis der Zellanpassung an räumliche Einschränkung
beitragen konnte. / Proper cell shape is a precondition for the proper performance of specialized cells and changes of cell shape are paramount for the development from a cell cluster to an adult organism. Cell shape can be regulated biochemically and also biophysically, e. g., by involvement of cellular force generation and spatial confinement. However, the understanding of the interaction between exterior space, cellular form, and function is incomplete. Therefore, the aim of this thesis was to thoroughly characterize cells in spatial confinement in order to better understand how cell morphology and force generation can be linked.
During the course of this work, the adaptation of human primary cells (HUVECs) to lateral constraints was investigated. Cells were seeded on both glass and hydrogel substrates which had been micropatterned with fibronectin by microcontact printing. The structures were composed of stripes with varying width (5–80 μm). After initial adhesion had taken place (> 1 h), cell morphology, actin cytoskeleton architecture, and cell traction forces were quantified. In addition, measurements were performed on live cells in order to better understand the dynamics of the actin cytoskeleton and the cell traction forces.
Laterally confined cells showed both structural and functional changes. Because cell length was only weakly dependent on stripe width, cells in strong lateral confinement were highly elongated and had decreased spread areas, which also affected the nucleus. The orientation of actin fibers was strongly linked to cell elongation. In cells on narrow stripes, a reduced actin cytoskeleton was observed, i.e., with a lower degree of interconnectivity. Time resolved analysis revealed an inward transport of actin fibers. Furthermore, cell force generation was shown to be impaired on narrow stripes, most likely due to decreased cell spread area. At the same time, force polarization strongly increased in cells in strong lateral confinement. This study demonstrated how various cellular parameters, both linked
to cell structure and function, are influenced by lateral confinement and by each other, thereby contributing to a better understanding of cell adaptation to spatial constraint.
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