Cell-generated Forces Research Articles

From static to animated: Measuring mechanical forces in tissues.

Cells are physical objects that exert mechanical forces on their surroundings as they migrate and take their places within tissues. New techniques are now poised to enable the measurement of cell-generated mechanical forces in intact tissues in vivo, which will illuminate the secret dynamic lives of cells and change our current perception of cell biology.

Cell sheet mechanics: How geometrical constraints induce the detachment of cell sheets from concave surfaces.

Despite of the significant progress in 3D tissue fabrication technologies at the microscale, there is still no quantitative model that can predict if cells seeded on a 3D structure maintain the imposed geometry while they form a continuous microtissue. Especially, detachment or loss of shape control of growing tissue is a major concern when designing 3D-structured scaffolds. Utilizing semi-cylindrical channels and vascular smooth muscle cells, we characterized how geometrical and mechanical parameters such as curvature of the substrate, cellular contractility, or protein-substrate adhesion strength tune the catastrophic detachment of microtissue. Observed results were rationalized by a theoretical model. The phase diagram showing how unintended tissue detachment progresses would help in designing of mechanically-balanced 3D scaffolds in future tissue engineering applications.

Measuring cell-generated forces: a guide to the available tools.

Forces generated by cells are critical regulators of cell adhesion, signaling, and function, and they are also essential drivers in the morphogenetic events of development. Over the past 20 years, several methods have been developed to measure these forces. However, despite recent substantial interest in understanding the contribution of these forces in biology, implementation and adoption of the developed methods by the broader biological community remain challenging because of the inherently multidisciplinary expertise required to conduct and interpret the measurements. In this review, we introduce the established methods and highlight the technical challenges associated with implementing each technique in a biological laboratory.

A Computational Model of Cell-Generated Traction Forces and Fibronectin Assembly

The extracellular matrix (ECM) is an assembly of proteins that surround cells and serves as the cell substrate in vivo. A primary component of newly synthesized ECM is fibronectin (FN), critical for embryonic development and wound healing. Despite years of research, the mechanism of FN assembly is still not understood. We hypothesize that FN assembly occurs through the revelation of buried FN-FN binding sites within any of the15 Type III FN domains, and that these binding sites are exposed by stretching of FN-III beta strands, allowing attachment of additional FN molecules via a beta strand addition mechanism. To investigate this hypothesis, we developed a biophysical computational model of cell-FN-substrate biomechanical-chemical interactions. In the model, FN-III domains are represented by Hookean springs with distinct stiffnesses; thus, each FN dimer is represented by 30 springs in series. Integrin binding/unbinding is represented by a stochastic first-order reversible chemical reaction with force-dependent off-rate. Model results are validated using in vitro tools: FN fibril growth and traction forces applied over time are measured using microfabricated pillar arrays in a human fibroblast cell line. Beta-strand exposure is probed using Thioflavin T, a fluorescent probe that binds to free beta strands. Results indicate that the computational model recapitulates three unique features that are observed in experimental measurements of FN fibrils: 1) FN fibrils are highly elastic, but have a maximal 4-fold elongation of their resting length; 2) FN fibril length and force are not strongly correlated; and 3) FN fibrils have discrete stable lengths, suggesting local minima of force balance within growing fibrils. These results will be discussed and compared with simulations in which FN fibril assembly is mediated by either a single cryptic binding site or a subset of cryptic binding sites.

Studying Cell Dynamics using Quantitative Phase Imaging

Most living cells do not absorb or scatter light significantly, i.e. they are essentially transparent, or phase objects. Phase contrast microscopy proposed by Zernike in the 1930's represents a major advance in intrinsic contrast imaging, as it reveals inner details of transparent structures without staining or tagging. While phase contrast is sensitive to minute optical path-length changes in the cell, down to the nanoscale, the information retrieved is only qualitative. Quantifying cell-induced shifts in the optical path-lengths permits nanometer scale measurements of structures and motions in a non-contact, non-invasive manner. Thus, quantitative phase imaging (QPI) has recently become an active field of study and various experimental approaches have been proposed.Recently, we have developed Spatial Light Interference microscopy (SLIM) as a highly sensitive QPI method. Due to its sub-nanometer pathlength sensitivity, SLIM enables interesting structure and dynamics studies over broad spatial (nanometers-centimeters) and temporal (milliseconds-weeks) scales. I will review our recent results on applying SLIM to basic cell studies, such as intracellular transport and cell growth. I will end with a discussion on new method, inspired from interferometry, for measuring cell-generated forces.

On Buckling Morphogenesis.

Cell-generated mechanical forces drive many of the tissue movements and rearrangements that are required to transform simple populations of cells into the complex three-dimensional geometries of mature organs. However, mechanical forces do not need to arise from active cellular movements. Recent studies have illuminated the roles of passive forces that result from mechanical instabilities between epithelial tissues and their surroundings. These mechanical instabilities cause essentially one-dimensional epithelial tubes and two-dimensional epithelial sheets to buckle or wrinkle into complex topologies containing loops, folds, and undulations in organs as diverse as the brain, the intestine, and the lung. Here, I highlight examples of buckling and wrinkling morphogenesis, and suggest that this morphogenetic mechanism may be broadly responsible for sculpting organ form.

Engineering 3D muscle microtissues by co-culturing human myoblasts and fibroblasts

Event Abstract Back to Event Engineering 3D muscle microtissues by co-culturing human myoblasts and fibroblasts Benoît Kalman1, 2, Claire Monge1, 2, Catherine Picart1, 2 and Thomas Boudou1, 2 1 CNRS UMR 5628 (LMGP), France 2 Université Grenoble Alpes - Grenoble Institute of Technology, France Introduction: Actual in vitro models for skeletal muscle tissue engineering present many limitations for high content screening analysis: 2D models do not mimic the native cellular microenvironment[1], whereas 3D models require large amounts of cells, thus limiting the reproducibility of experiments[2],[3]. In our lab, we developed a microfabricated platform for generating arrays of 3D microtissues with force tracking in real time[4]. Here, we demonstrate how tissue formation, stability, and cell differentiation in microtissues generated from immortalized human myoblasts can be improved by co-culturing the myoblasts with fibroblasts. Materials and Methods: Human immortalized myoblasts were kindly provided by the Institute of Myology (Paris, France). Human fibroblasts BJ-5ta were obtained from ATCC. Polydimethylsiloxane (PDMS) platforms were made by casting Sylgard 184 liquid prepolymer over a silicon master fabricated by UV photolithography. The platforms consisted of 800x400x160µm wells containing two T-shape microcantilevers (Fig. 1.A). A cooled suspension of cells within a reconstitution mixture consisting of collagen I and matrigel was then added to the platform, resulting in around 200 cells per well, before incubation at 37 °C to induce collagen/matrigel polymerization. Cell-generated forces were quantified from the deflection of the microcantilevers. Fig. 1: Engineering of human muscle microtissues. (A) Schematic of the microfabrication of the platform. (B) Temporal evolution of muscle microtissues constructed in collagen/matrigel gels in our microfabricated platform. The scale bar is 200µm. (C) Effect of human myoblasts/fibroblasts co-culture on the stability of microtissues after 3 days in culture. Results and discussion: After cell seeding, the collagen/matrigel matrix contained evenly distributed round cells. Over time, cells elongated and compacted the matrix to form a microtissue spanning between the two microcantilevers (Fig. 1.B). After 3 days in culture, we observed that micromuscles composed only of human myoblasts tended to rupture (only 46% of stable tissues) whereas micromuscles consisting of 20% fibroblasts and 80% human myoblasts remained stable over time (94% of stable tissues) (Fig. 1.C). After 3 days in culture, the tension generated by muscle microtissues composed only of human myoblasts reached 20 µN while tissues composed of 20% fibroblasts and 80% human myoblasts generated a tension of 13 µN. We posit that fibroblasts helped stabilizing muscle microtissues by lowering the tissue-generated tension and by secreting additional extracellular matrix, thus reinforcing the integrity of the tissue. Interestingly, further analysis showed that fibroblasts improved spontaneous myoblasts differentiation toward myotubes after 5 days in culture. Conclusions: In this work we showed that engineered microtissues from a co-culture of human myoblasts and myoblasts exhibited a more stable morphology, thanks to a reinforcement of the extracellular matrix secreted by fibroblasts. The degree of differentiation of the muscle microtissues was also improved by the co-culture. The overall similarity of structural and functional characteristics between muscle microtissues and in vivo skeletal muscle is promising and our novel method for micromuscle generation opens the potential to high throughput, low volume screening applications. Moreover, the model’s ability to quantitatively demonstrate the impact of biological and physical parameters on the formation, maturation, structure and function of muscle tissue provide unique opportunities to elucidate mechanisms of myogenesis in stable, three-dimensional, working muscle preparations. The authors thank the members of the technical staff of the PTA cleanroom in Grenoble for their technical support; We acknowledge the platform for immortalization of human cells from the Institute of Myology for providing us with human immortalized myoblasts; This work was supported by the European Commission (under FP7) via an ERC starting grant (BIOMIM, GA 259370) and by the Association Française contre les Myopathies (AFM-Telethon, project n°316530). CM is indebted to AFM-Telethon for providing a post-doctoral fellowship (n°16673)

Myofibroblast persistence with real-time changes in boundary stiffness

Myofibroblasts are critical for connective tissue remodeling and wound healing since they can close wound beds and shape tissues rapidly by generating high traction forces and secreting abundant extracellular matrix proteins and matrix metalloproteinases. However, their presence in excessive numbers is associated with fibrotic and calcific diseases and tissue thickening in engineered tissues. While activation of the myofibroblast phenotype has been studied extensively, whether myofibroblasts are “cleared” by phenotypic reversal or by apoptosis remains controversial. The goal of this work is to test the hypothesis that mechanical inhibition of myofibroblast force generation leads to de-differentiation or apoptosis depending upon the magnitude of the decrease in tension. To test this hypothesis, we cultured valvular interstitial cells (VICs) in fibrin micro-tissues suspended between flexible posts and dynamically altered the ability of the cells to generate tension by altering boundary stiffness via magnetic forces applied to posts. The flexible posts capped with magnetic beads enable the measurement and modulation of tension generated by the cells within the tissue. As expected, the cell-generated forces were elevated with dynamically increased boundary (post) stiffness, yet surprisingly, the forces continued to increase following dynamic reduction of boundary stiffness back to baseline levels. Increased apoptosis and reduced α-SMA staining were observed with complete freeing of the tissues from the posts but not upon removal of the magnet, resulting in a twofold decrease in post stiffness. Together, these data indicate that an increase in myofibroblast force generation, even if modest and temporary (1day), can have lasting effects on myofibroblast persistence in tissues, and that a significant reduction in the ability of the cells to generate tension is required to trigger dedifferentiation and/or apoptosis. The ability to dedifferentiate myofibroblasts to a quiescent phenotype and to control the percentage of apoptosis would be of great benefit for therapeutic and tissue engineering applications. Statement of SignificanceMyofibroblasts play an important role in tissue remodeling and wound healing. However, excessive activation of this phenotype is associated with fibrotic diseases and scar formation. Being able to dedifferentiate these cells or controlling their clearance with apoptosis (programmed cell death) would be beneficial. It is known that releasing rigid tissue boundaries trigger apoptosis, while reducing the substrate stiffness can cause myofibroblast dedifferentiation. However, the mechanical tension was not quantified in any of the studies. Here we used micro-cantilever posts at tissue boundaries to measure tension and to regulate boundary stiffness in real time by pulling posts with magnets. We show that temporary stiffening of boundary causes irreversible myofibroblast activation and the magnitude of tension drop controls apoptosis.

Three-dimensional force microscopy of cells in biopolymer networks.

We describe a technique for the quantitative measurement of cell-generated forces in highly nonlinear three-dimensional biopolymer networks that mimic the physiological situation of living cells. We computed forces of MDA-MB-231 breast carcinoma cells from the measured network deformations around the cells using a finite-element approach based on a constitutive equation that captures the complex mechanical properties of diverse biopolymers such as collagen gels, fibrin gels and Matrigel. Our measurements show that breast carcinoma cells cultured in collagen gels generated nearly constant forces regardless of the collagen concentration and matrix stiffness. Furthermore, time-lapse force measurements showed that these cells migrated in a gliding motion with alternating phases of high and low contractility, elongation, migratory speed and persistence.

Fibronectin fibrillogenesis facilitates mechano-dependent cell spreading, force generation, and nuclear size in human embryonic fibroblasts.

Cells respond to mechanical cues from the substrate to which they are attached. These mechanical cues drive cell migration, proliferation, differentiation, and survival. Previous studies have highlighted three specific mechanisms through which substrate stiffness directly alters cell function: increasing stiffness drives (1) larger contractile forces; (2) increased cell spreading and size; and (3) altered nuclear deformation. While studies have shown that substrate mechanics are an important cue, the role of the extracellular matrix (ECM) has largely been ignored. The ECM is a crucial component of the mechanosensing system for two reasons: (1) many ECM fibrils are assembled by application of cell-generated forces, and (2) ECM proteins have unique mechanical properties that will undoubtedly alter the local stiffness sensed by a cell. We specifically focused on the role of the ECM protein fibronectin (FN), which plays a critical role in de novo tissue production. In this study, we first measured the effects of substrate stiffness on human embryonic fibroblasts by plating cells onto microfabricated pillar arrays (MPAs) of varying stiffness. Cells responded to increasing substrate stiffness by generating larger forces, spreading to larger sizes, and altering nuclear geometry. These cells also assembled FN fibrils across all stiffnesses, with optimal assembly occurring at approximately 6 kPa. We then inhibited FN assembly, which resulted in dramatic reductions in contractile force generation, cell spreading, and nuclear geometry across all stiffnesses. These findings suggest that FN fibrils play a critical role in facilitating cellular responses to substrate stiffness.

Spatial distribution of cell-cell and cell-ECM adhesions regulates force balance while main-taining E-cadherin molecular tension in cell pairs.

Mechanical linkage between cell-cell and cell-extracellular matrix (ECM) adhesions regulates cell shape changes during embryonic development and tissue homoeostasis. We examined how the force balance between cell-cell and cell-ECM adhesions changes with cell spread area and aspect ratio in pairs of MDCK cells. We used ECM micropatterning to drive different cytoskeleton strain energy states and cell-generated traction forces and used a Förster resonance energy transfer tension biosensor to ask whether changes in forces across cell-cell junctions correlated with E-cadherin molecular tension. We found that continuous peripheral ECM adhesions resulted in increased cell-cell and cell-ECM forces with increasing spread area. In contrast, confining ECM adhesions to the distal ends of cell-cell pairs resulted in shorter junction lengths and constant cell-cell forces. Of interest, each cell within a cell pair generated higher strain energies than isolated single cells of the same spread area. Surprisingly, E-cadherin molecular tension remained constant regardless of changes in cell-cell forces and was evenly distributed along cell-cell junctions independent of cell spread area and total traction forces. Taken together, our results showed that cell pairs maintained constant E-cadherin molecular tension and regulated total forces relative to cell spread area and shape but independently of total focal adhesion area.

Structural biology response of a collagen hydrogel synthetic extracellular matrix with embedded human fibroblast: computational and experimental analysis

Adherent cells exert contractile forces which play an important role in the spatial organization of the extracellular matrix (ECM). Due to these forces, the substrate experiments a volume reduction leading to a characteristic shape. ECM contraction is a key process in many biological processes such as embryogenesis, morphogenesis and wound healing. However, little is known about the specific parameters that control this process. With this aim, we present a 3D computational model able to predict the contraction process of a hydrogel matrix due to cell-substrate mechanical interaction. It considers cell-generated forces, substrate deformation, ECM density, cellular migration and proliferation. The model also predicts the cellular spatial distribution and concentration needed to reproduce the contraction process and confirms the minimum value of cellular concentration necessary to initiate the process observed experimentally. The obtained continuum formulation has been implemented in a finite element framework. In parallel, in vitro experiments have been performed to obtain the main model parameters and to validate it. The results demonstrate that cellular forces, migration and proliferation are acting simultaneously to display the ECM contraction.

Mechanics of epithelial closure over non-adherent environments.

The closure of gaps within epithelia is crucial to maintain its integrity during biological processes such as wound healing and gastrulation. Depending on the distribution of extracellular matrix, gap closure occurs through assembly of multicellular actin-based contractile cables or protrusive activity of border cells into the gap. Here we show that the supracellular actomyosin contractility of cells near the gap edge exerts sufficient tension on the surrounding tissue to promote closure of non-adherent gaps. Using traction force microscopy, we observe that cell-generated forces on the substrate at the gap edge first point away from the centre of the gap and then increase in the radial direction pointing into the gap as closure proceeds. Combining with numerical simulations, we show that the increase in force relies less on localized purse-string contractility and more on large-scale remodelling of the suspended tissue around the gap. Our results provide a framework for understanding the assembly and the mechanics of cellular contractility at the tissue level.

Direct laser manipulation reveals the mechanics of cell contacts in vivo

Cell-generated forces produce a variety of tissue movements and tissue shape changes. The cytoskeletal elements that underlie these dynamics act at cell-cell and cell-ECM contacts to apply local forces on adhesive structures. In epithelia, force imbalance at cell contacts induces cell shape changes, such as apical constriction or polarized junction remodeling, driving tissue morphogenesis. The dynamics of these processes are well-characterized; however, the mechanical basis of cell shape changes is largely unknown because of a lack of mechanical measurements in vivo. We have developed an approach combining optical tweezers with light-sheet microscopy to probe the mechanical properties of epithelial cell junctions in the early Drosophila embryo. We show that optical trapping can efficiently deform cell-cell interfaces and measure tension at cell junctions, which is on the order of 100 pN. We show that tension at cell junctions equilibrates over a few seconds, a short timescale compared with the contractile events that drive morphogenetic movements. We also show that tension increases along cell interfaces during early tissue morphogenesis and becomes anisotropic as cells intercalate during germ-band extension. By performing pull-and-release experiments, we identify time-dependent properties of junctional mechanics consistent with a simple viscoelastic model. Integrating this constitutive law into a tissue-scale model, we predict quantitatively how local deformations propagate throughout the tissue.

Mechanoregulation of aortic valvular interstitial cell life and death.

Valvular interstitial cells (VICs) are the major cell type within aortic valve leaflets. VICs are able to exhibit a spectrum of phenotype characteristics including those of fibroblasts, smooth muscle cells, and myofibroblasts. VICs are responsible for valve maintenance and repair, yet excessive persistence of the myofibroblast phenotype is implicated in a number of valve diseases, including calcific aortic valve disease and fibrosis. Despite the prevalence of these diseases, the stimuli regulating the transition to the activated myofibroblast state and reversal to quiescent fibroblast and/or induction of apoptosis are not fully understood. The purpose of this article is to review in vitro studies that have contributed to the current understanding of mechanical regulation of VIC phenotype and fate. In particular, we have focused on studies utilizing advanced in vitro systems that allow modulation and measurement of cell tension and cell-generated forces in two-dimensional and three-dimensional cultures. In addition, we discuss the importance of cell tension in phenotype modulation and how cytoskeletal tension may contribute to aggregation and calcification. Future directions of pharmaceutical development aimed at reducing VIC cytoskeletal tension are also highlighted.

A Computational Model of Cell-Generated Traction Forces and Fibronectin Assembly

A Computational Model of Cell-Generated Traction Forces and Fibronectin Assembly

Displacement of p130Cas from focal adhesions links actomyosin contraction to cell migration

Cell adhesion complexes provide platforms where cell-generated forces are transmitted to the extracellular matrix (ECM). Tyrosine phosphorylation of focal adhesion proteins is crucial for cells to communicate with the extracellular environment. However, the mechanisms that transmit actin cytoskeletal motion to the extracellular environment to drive cell migration are poorly understood. We find that the movement of p130Cas (Cas, also known as BCAR1), a mechanosensor at focal adhesions, correlates with actin retrograde flow and depends upon actomyosin contraction and phosphorylation of the Cas substrate domain (CasSD). This indicates that CasSD phosphorylation underpins the physical link between Cas and the actin cytoskeleton. Fluorescence recovery after photobleaching (FRAP) experiments reveal that CasSD phosphorylation, as opposed to the association of Cas with Src, facilitates Cas displacement from adhesion complexes in migrating cells. Furthermore, the stabilization of Src-Cas binding and inhibition of myosin II, both of which sustain CasSD phosphorylation but mitigate Cas displacement from adhesion sites, retard cell migration. These results indicate that Cas promotes cell migration by linking actomyosin contractions to the adhesion complexes through a dynamic interaction with Src as well as through the phosphorylation-dependent association with the actin cytoskeleton.

Protrusive Activity Guides Changes in Cell-Cell Tension during Epithelial Cell Scattering

Knowing how epithelial cells regulate cell-matrix and cell-cell adhesions is essential to understand key events in morphogenesis as well as pathological events such as metastasis. During epithelial cell scattering, epithelial cell islands rupture their cell-cell contacts and migrate away as single cells on the extracellular matrix (ECM) within hours of growth factor stimulation, even as adhesion molecules such as E-cadherin are present at the cell-cell contact. How the stability of cell-cell contacts is modulated to effect such morphological transitions is still unclear. Here, we report that in the absence of ECM, E-cadherin adhesions continue to sustain substantial cell-generated forces upon hepatocyte growth factor (HGF) stimulation, consistent with undiminished adhesion strength. In the presence of focal adhesions, constraints that preclude the spreading and movement of cells at free island edges also prevent HGF-mediated contact rupture. To explore the role of cell motion and cell-cell contact rupture, we examine the biophysical changes that occur during the scattering of cell pairs. We show that the direction of cell movement with respect to the cell-cell contact is correlated with changes in the average intercellular force as well as the initial direction of cell-cell contact rupture. Our results suggest an important role for protrusive activity resulting in cell displacement and force redistribution in guiding cell-cell contact rupture during scattering.

Cell-generated traction forces and the resulting matrix deformation modulate microvascular alignment and growth during angiogenesis.

The details of the mechanical factors that modulate angiogenesis remain poorly understood. Previous in vitro studies of angiogenesis using microvessel fragments cultured within collagen constructs demonstrated that neovessel alignment can be induced via mechanical constraint of the boundaries (i.e., boundary conditions). The objective of this study was to investigate the role of mechanical boundary conditions in the regulation of angiogenic alignment and growth in an in vitro model of angiogenesis. Angiogenic microvessels within three-dimensional constructs were subjected to different boundary conditions, thus producing different stress and strain fields during growth. Neovessel outgrowth and orientation were quantified from confocal image data after 6 days. Vascularity and branching decreased as the amount of constraint imposed on the culture increased. In long-axis constrained hexahedral constructs, microvessels aligned parallel to the constrained axis. In contrast, constructs that were constrained along the short axis had random microvessel orientation. Finite element models were used to simulate the contraction of gels under the various boundary conditions and to predict the local strain field experienced by microvessels. Results from the experiments and simulations demonstrated that microvessels aligned perpendicular to directions of compressive strain. Alignment was due to anisotropic deformation of the matrix from cell-generated traction forces interacting with the mechanical boundary conditions. These findings demonstrate that boundary conditions and thus the effective stiffness of the matrix regulate angiogenesis. This study offers a potential explanation for the oriented vascular beds that occur in native tissues and provides the basis for improved control of tissue vascularization in both native tissues and tissue-engineered constructs.

Force-driven evolution of mesoscale structure in engineered 3D microtissues and the modulation of tissue stiffening

The complex structures of tissues determine their mechanical strength. In engineered tissues formed through self-assembly in a mold, artificially imposed boundary constraints have been found to induce anisotropic clustering of the cells and the extracellular matrix in local regions. To understand how such tissue remodeling at the intermediate length-scale (mesoscale) affects tissue stiffening, we used a novel microtissue mechanical testing system to manipulate the remodeling of the tissue structures and to measure the subsequent changes in tissue stiffness. Microtissues were formed through cell driven self-assembly of collagen matrix in arrays of micro-patterned wells, each containing two flexible micropillars that measured the microtissues' contractile forces and elastic moduli via magnetic actuation. We manipulated tissue remodeling by inducing myofibroblast differentiation with TGF-β1, by varying the micropillar spring constants or by blocking cell contractility with blebbistatin and collagen cross-linking with BAPN. We showed that increased anisotropic compaction of the collagen matrix, caused by increased micropillar spring constant or elevated cell contraction force, contributed to tissue stiffening. Conversely, collagen matrix and tissue stiffness were not affected by inhibition of cell-generated contraction forces. Together, these measurements showed that mesoscale tissue remodeling is an important middle step linking tissue compaction forces and tissue stiffening.