"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."

"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."

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.

Cell adhesion to extracellular matrix (ECM) is critical to various cellular processes like cell spreading, migration, growth and apoptosis. At the tissue level, cell adhesion is important in the pathological and physiological processes that regulate the tissue morphogenesis. Cell adhesion to the ECM is primarily mediated by the integrin family of receptors. The receptors that are recruited to the surface are reinforced by structural and signaling proteins at the adhesive sites forming focal adhesions that connect the cytoskeleton to further stabilize the adhesions. The functional roles of these focal adhesions extend beyond stabilizing adhesions and transduce mechanical signals at the cell-ECM interface in various signaling events. The objective of this research is to analyze the role of the spatial distribution of the focal adhesions in stabilizing the cell adhesion to the ECM in relation to cell's internal force balance. The central hypothesis was that peripheral focal adhesions stabilize cell adhesion to ECM by providing for maximum mechanical advantage for resisting detachment as explained by the membrane peeling mechanism. Micropatterning techniques combined with robust hydrodynamic shear assay were employed to test our hypothesis. However, technical difficulties in microcontact printing stamps with small and sparse features made it challenging to analyze the role of peripheral focal adhesions in stabilizing cell adhesion. To overcome this limitation, the roof collapse phenomenon in stamps with small and sparse features (low fill factor stamps) that was detrimental to the reproduction of the adhesive geometries required to test the hypothesis was analyzed. This analysis lead to the valuable insight that the non-uniform pressure distribution during initial contact caused by parallelism error during manual microcontact printing prevented accurate replication of features on the substrate. To this end, the template of the stamp was modified so that it included an annular column around the pattern zone that acted as a collapse barrier and prevented roof collapse propagation into the pattern zone. Employing this modified stamp, the required geometries for the cell adhesion analysis were successfully reproduced on the substrates with high throughput. Adhesive areas were engineered with circular and annular patterns to discern the contribution of peripheral focal adhesions towards cell adhesion strength. The patterns were engineered such that two distinct geometries with either constant adhesive area or constant spreading area were obtained. The significance of annular patterns is that for the same total adhesive area as the circular pattern, the annular pattern provided for greater cell spreading thereby increasing the distance of the focal adhesions from the cell's center. The adhesion strength analysis was accomplished by utilizing hydrodynamic shear flow in a spinning disk device that was previously developed. The results indicate that for a constant total adhesive area, the annular patterns provide for greater adhesion strength by enhancing cell spreading area and providing for greater moment arm in resisting detachment due to shear. The next examination was the effect of the cell's internal force balance in stabilizing the cell adhesion. The working hypothesis was that microtubules provide the necessary forces to resist the tensile forces expressed by the cell contractile machinery, thereby stabilizing cell adhesion. Since microtubule disruption is known to enhance cell contractility, its effect on the cell adhesion strength was examined. Moreover, the force balance in cells was altered by engineering adhesive areas so that the cells were either spherical or completely spread and then disrupted microtubules to understand the significance of the force balance in modulating the cell adhesion strength. The results indicated that disruption of microtubules in cells on adhesive islands resulted in a 10 fold decrease in adhesion strength compared to untreated controls whereas no significant change was observed in completely spread cells between treated and untreated controls. This is in surprising contrast to the previous contractility inhibition studies which indicate a less pronounced regulation of adhesion strength for both micropatterned and spread cells. Taken together, these findings suggest that the internal force balance regulated by cell shape strongly modulates the adhesion strength though the microtubule network. In summary, this project elucidates the role of peripheral focal adhesions in regulating the cell adhesion strength. Furthermore, this study also establishes the importance of the internal force balance towards stabilizing the cell adhesion to the ECM through the microtubule network.

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.

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

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.

Die Untersuchung von Zell-Material-Wechselwirkungen ist bedeutsam für die Entwicklung innovativer Biomaterialien, wobei aus biophysikalischer Sicht der Einfluss mechanischer Eigenschaften auf das Zellverhalten, d.h., die Mechanotransduktion, von besonderem Interesse ist. Für diese Dissertation wurden humane Endothelzellen aus der Nabelschnurvene zur Adhäsion auf Polyacrylamidhydrogele (PAA-Hydrogele) gegeben, die mit einer Maleinsäurecopolymer-Beschichtung versehen waren. Für Experimente unter veränderlichen Substrateigenschaften wurden die Steifigkeit der PAA-Hydrogele und die Ligandenaffinität der Beschichtung variiert. Der erste Teil der Dissertation umfasste die Charakterisierung der beschichteten PAA-Hydrogele. Dafür wurde der Elastizitätsmodul gemessen und die Adsorption von Fibronektin untersucht. Im zweiten Teil der Dissertation wurden die PAA-Hydrogele in der Zellzugkraftmikroskopie während der initialen Zelladhäsion (2 h) verwendet. Dabei stellte sich heraus, dass zwar die finale Zellfläche unabhängig von den Substratparametern war, aber die Ausbreitung von Zellen mit zunehmender Steifigkeit und Ligandenaffinität schneller ablief. Außerdem waren der Anstieg und die Plateauwerte der Zellzugkräfte auf steiferen Substraten größer. Die Steifigkeitsabhängigkeit lässt sich aus der Dehnungsversteifung des Aktinzytoskeletts unter Wirkung einer Spannung erklären. Eine Zunahme der Ligandenaffinität führte ebenfalls zu einer schnelleren Zunahme und größeren Plateauwerten von Gesamtzellzugkräften. Diese Beobachtung kann der Zunahme von Reibungskräften zugesprochen werden. Im letzten Teil der Dissertation sollten die biophysikalischen Ergebnisse durch die Untersuchung intrazellulärer Signalprozesse zusätzlich unterlegt werden. Dafür wurde die Entwicklung von Adhäsionsstellen durch eine immunzytochemische Färbung untersucht. Obwohl diese aufgrund der technischen Herausforderungen keine umfassenden Aussagen liefern konnte, deuteten sich einige Korrelationen, z.B. eine schnellere Entwicklung der Adhäsionsstellen auf steiferen Substraten, an. Die Ergebnisse der Dissertation ordnen sich in den aktuellen Forschungsstand zur Mechanotransduktion von Zellen ein und konnten in Bezug auf die Adhäsionsdynamik neue Erkenntnisse beisteuern. Vor allem der Stellenwert dissipativer Beiträge zu Zell-Substrat-Wechselwirkungen (z.B. Ligandenreibung) wurde unterstrichen. Diese sind in der Entwicklung neuer Biomaterialien mit spezifischen viskoelastischen Eigenschaften von besonderer Bedeutung. / The investigation of cell-substrate-interactions is of great importance for the development of innovative biomaterials. The influence of thematerials mechanical properties on cells and their functions, i. e., the process of mechanotransduction, is of particular interest from a biophysical point of view. In this dissertation human umbilical cord vein endothelial cells were seeded onto polyacrylamide hydrogels which had been modified by a maleic acid copolymer coating. To tune the mechanical properties of the substrate the hydrogels’ stiffness and the affinity of the coatings to the adhesion ligand fibronectin were variied. The first part of the dissertation is concerned with the characterization of the coated polyacrylamide hydrogels. The hydrogels’ Young’s modulus was measured and the adsorption of fibronectin was investigated. In the second part of the dissertation these cell culture scaffolds were used for cell traction force microscopy during the first two hours of cell adhesion. Although maximum cell area was not influenced by substrate parameters, cell spreading was faster for higher stiffness and higher ligand affinity. Traction force increase as well as plateau forces were higher on stiff substrates. The dependence of the dynamics of area and traction force on stiffness and their respective magnitudes after saturation could be related to properties of the actin cytoskeleton under stress. The increase in ligand affinity also led to a faster increase and higher mean plateau values of the total cell force. This observation was assigned to increasing friction forces between ligands and polymer coating. In the last part of the dissertation possible correlations between cell traction forces and intracellular signalling processes were examined. The development of adhesion sites during early cell adhesion was investigated by immunocytochemical staining. Due to technical reasons no comprehensive investigation could be realized, but nevertheless some correlations were observed, such as a faster adhesion site formation with higher stiffness. The results of this dissertation add to the current state of research regarding mechanotransduction of cells and yield new findings regarding to cell adhesion dynamics. Most notably viscous contributions to cell-substrate-interactions (i.e., ligand friction) were shown to influence cell behavior. This highlights that a thorough understanding of viscous processes is of utmost significance for the development of new biomaterials with specific viscoelastic properties.

Die Untersuchung von Zell-Material-Wechselwirkungen ist bedeutsam für die Entwicklung innovativer Biomaterialien, wobei aus biophysikalischer Sicht der Einfluss mechanischer Eigenschaften auf das Zellverhalten, d.h., die Mechanotransduktion, von besonderem Interesse ist. Für diese Dissertation wurden humane Endothelzellen aus der Nabelschnurvene zur Adhäsion auf Polyacrylamidhydrogele (PAA-Hydrogele) gegeben, die mit einer Maleinsäurecopolymer-Beschichtung versehen waren. Für Experimente unter veränderlichen Substrateigenschaften wurden die Steifigkeit der PAA-Hydrogele und die Ligandenaffinität der Beschichtung variiert. Der erste Teil der Dissertation umfasste die Charakterisierung der beschichteten PAA-Hydrogele. Dafür wurde der Elastizitätsmodul gemessen und die Adsorption von Fibronektin untersucht. Im zweiten Teil der Dissertation wurden die PAA-Hydrogele in der Zellzugkraftmikroskopie während der initialen Zelladhäsion (2 h) verwendet. Dabei stellte sich heraus, dass zwar die finale Zellfläche unabhängig von den Substratparametern war, aber die Ausbreitung von Zellen mit zunehmender Steifigkeit und Ligandenaffinität schneller ablief. Außerdem waren der Anstieg und die Plateauwerte der Zellzugkräfte auf steiferen Substraten größer. Die Steifigkeitsabhängigkeit lässt sich aus der Dehnungsversteifung des Aktinzytoskeletts unter Wirkung einer Spannung erklären. Eine Zunahme der Ligandenaffinität führte ebenfalls zu einer schnelleren Zunahme und größeren Plateauwerten von Gesamtzellzugkräften. Diese Beobachtung kann der Zunahme von Reibungskräften zugesprochen werden. Im letzten Teil der Dissertation sollten die biophysikalischen Ergebnisse durch die Untersuchung intrazellulärer Signalprozesse zusätzlich unterlegt werden. Dafür wurde die Entwicklung von Adhäsionsstellen durch eine immunzytochemische Färbung untersucht. Obwohl diese aufgrund der technischen Herausforderungen keine umfassenden Aussagen liefern konnte, deuteten sich einige Korrelationen, z.B. eine schnellere Entwicklung der Adhäsionsstellen auf steiferen Substraten, an. Die Ergebnisse der Dissertation ordnen sich in den aktuellen Forschungsstand zur Mechanotransduktion von Zellen ein und konnten in Bezug auf die Adhäsionsdynamik neue Erkenntnisse beisteuern. Vor allem der Stellenwert dissipativer Beiträge zu Zell-Substrat-Wechselwirkungen (z.B. Ligandenreibung) wurde unterstrichen. Diese sind in der Entwicklung neuer Biomaterialien mit spezifischen viskoelastischen Eigenschaften von besonderer Bedeutung. / The investigation of cell-substrate-interactions is of great importance for the development of innovative biomaterials. The influence of thematerials mechanical properties on cells and their functions, i. e., the process of mechanotransduction, is of particular interest from a biophysical point of view. In this dissertation human umbilical cord vein endothelial cells were seeded onto polyacrylamide hydrogels which had been modified by a maleic acid copolymer coating. To tune the mechanical properties of the substrate the hydrogels’ stiffness and the affinity of the coatings to the adhesion ligand fibronectin were variied. The first part of the dissertation is concerned with the characterization of the coated polyacrylamide hydrogels. The hydrogels’ Young’s modulus was measured and the adsorption of fibronectin was investigated. In the second part of the dissertation these cell culture scaffolds were used for cell traction force microscopy during the first two hours of cell adhesion. Although maximum cell area was not influenced by substrate parameters, cell spreading was faster for higher stiffness and higher ligand affinity. Traction force increase as well as plateau forces were higher on stiff substrates. The dependence of the dynamics of area and traction force on stiffness and their respective magnitudes after saturation could be related to properties of the actin cytoskeleton under stress. The increase in ligand affinity also led to a faster increase and higher mean plateau values of the total cell force. This observation was assigned to increasing friction forces between ligands and polymer coating. In the last part of the dissertation possible correlations between cell traction forces and intracellular signalling processes were examined. The development of adhesion sites during early cell adhesion was investigated by immunocytochemical staining. Due to technical reasons no comprehensive investigation could be realized, but nevertheless some correlations were observed, such as a faster adhesion site formation with higher stiffness. The results of this dissertation add to the current state of research regarding mechanotransduction of cells and yield new findings regarding to cell adhesion dynamics. Most notably viscous contributions to cell-substrate-interactions (i.e., ligand friction) were shown to influence cell behavior. This highlights that a thorough understanding of viscous processes is of utmost significance for the development of new biomaterials with specific viscoelastic properties.

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.