A culture force monitor for measurement of contraction forces generated in human dermal fibroblast cultures: evidence for cell-matrix mechanical signalling

Eastwood, Mark; McGrouther, Duncan Angus; Brown, Robert A.

doi:10.1016/0304-4165(94)90040-x

Cited by 187 publications

(192 citation statements)

References 14 publications

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“…Briefly, with 1 ϫ 10 6 cells/ml collagen gel, we measured the force generated across the collagen lattice, by using a tensioning-culture force monitor that is capable of measuring the minute forces exerted by cells within a collagen lattice (Eastwood et al, 1994), Ͼ24 h as fibroblasts attach, spread, and migrate. In brief, a rectangular fibroblastseeded collagen gel was cast and floated in medium, tethered to two flotation bars on either end of the short edges, in turn attached to an anchor point at one end and a force transducer at the other.…”

Section: Fibroblast Populated Collagen Lattices (Fpcl)mentioning

confidence: 99%

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Endothelin-1 Promotes Myofibroblast Induction through the ETA Receptor via a rac/Phosphoinositide 3-Kinase/Akt-dependent Pathway and Is Essential for the Enhanced Contractile Phenotype of Fibrotic Fibroblasts

Chen

Denton

et al. 2004

MBoC

327

307

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The endothelins are a family of endothelium-derived peptides that possess a variety of functions, including vasoconstriction. Endothelin-1 (ET-1) is up-regulated during tissue repair and promotes myofibroblast contraction and migration, hence contributing to matrix remodeling during tissue repair. Here, we show that addition of ET-1 to normal lung fibroblasts induces expression of proteins that contribute to a contractile phenotype, including ␣-smooth muscle actin (␣-SMA), ezrin, moesin, and paxillin. We confirm that ET-1 enhances the ability of lung fibroblasts to contract extracellular matrix, a function essential for tissue repair, through induction of de novo protein synthesis. Blockade of the Akt/ phosphoinositide 3-kinase (PI3-kinase) pathway with LY294002 and wortmannin prevents the ability of ET-1 to induce ␣-SMA, ezrin, paxillin, and moesin and to promote matrix contraction. Dominant negative rac and Akt blocked the ability of ET-1 to promote formation of ␣-SMA stress fibers. Using specific ET-1 receptor inhibitors, we show that ET-1 induces collagen matrix contraction through the ETA, but not the ETB, receptor. Relative to normal pulmonary fibroblasts, fibroblasts cultured from scars of patients with the fibrotic disease systemic sclerosis (scleroderma) show enhanced ET-1 expression and binding. Systemic sclerosis lung fibroblasts show increased ability to contract a collagen matrix and elevated expression of the procontractile proteins ␣-SMA, ezrin, paxillin, and moesin, which are greatly reduced by antagonizing endogenous ET-1 signaling. Thus, blocking ET-1 or the PI3-kinase/Akt cascades might be beneficial in reducing scar formation in pulmonary fibrosis. INTRODUCTIONA complex histological and architectural structure is a prerequisite for effective lung function. In the lung, specialized structures, the alveoli, increase the surface area of the lung, allowing for efficient gas exchange. The maintenance of these specialized structures is in turn dependent on the underlying connective tissue, comprised principally of fibroblasts and extracellular matrix (ECM; for review, see Gadek et al., 1984), which is essential for the mechanical and structural integrity of the lung. As a response to environmental insults, or as a consequence of local inflammatory processes, structural damage to the lung can occur, resulting in a wound healing response. This response consists of an integrated series of biochemical, immunological, and structural changes that result in the de novo synthesis of a new epithelium, blood vessels, and connective tissue (Razzaque and Taguchi, 2003). The proper repair of connective tissue requires synthesis of new ECM components, such as collagen and fibronectin (Badylak, 2002). In addition, repair of connective tissue requires the proper reconstitution of its support function; that is, an appropriate tensile strength must be recreated. This tensile strength results from the remodeling of the newly formed ECM through a combination of cell locomotion and translocation of the flexible collage...

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Section: Fibroblast Populated Collagen Lattices (Fpcl)mentioning

confidence: 99%

“…Cell-generated tensional forces in the collagen gel were detected by the force transducer and logged into a personal computer. Graphical readings are produced every 10 min, averaged from 600 readings (1/s), providing a continuous output of force (Dynes) generated (Eastwood et al, 1994). …”

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confidence: 99%

Endothelin-1 Promotes Myofibroblast Induction through the ETA Receptor via a rac/Phosphoinositide 3-Kinase/Akt-dependent Pathway and Is Essential for the Enhanced Contractile Phenotype of Fibrotic Fibroblasts

Chen

Denton

et al. 2004

MBoC

327

307

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“…To improve the accuracy of CGCA force measurements, a culture force monitor (CFM) system was developed by attaching strain gauges at the edges of a cell-embedded collagen gel to directly assess cell contraction [15][16][17][18][19][20]. Cellular contraction force can be measured continuously without influence of the elastic variation of collagen gel.…”

Section: Introductionmentioning

confidence: 99%

A novel collagen gel-based measurement technique for quantitation of cell contraction force

Jin

Siow

et al. 2015

J. R. Soc. Interface.

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ResearchCite this article: Jin T, Li L, Siow RCM, Liu K-K. 2015 A novel collagen gel-based measurement technique for quantitation of cell contraction force. J. R. Soc. Cell contraction force plays an important role in wound healing, inflammation, angiogenesis and metastasis. This study describes a novel method to quantify single cell contraction force in vitro using human aortic adventitial fibroblasts embedded in a collagen gel. The technique is based on a depth sensing nano-indentation tester to measure the thickness and elasticity of collagen gels containing stimulated fibroblasts and a microscopy imaging system to estimate the gel area. In parallel, a simple theoretical model has been developed to calculate cell contraction force based on the measured parameters. Histamine (100 mM) was used to stimulate fibroblast contraction while the myosin light chain kinase inhibitor ML-7 (25 mM) was used to inhibit cell contraction. The collagen matrix used in the model provides a physiological environment for fibroblast contraction studies. Measurement of changes in collagen gel elasticity and thickness arising from histamine treatments provides a novel convenient technique to measure cell contraction force within a collagen matrix. This study demonstrates that histamine can elicit a significant increase in contraction force of fibroblasts embedded in collagen, while the Young's modulus of the gel decreases due to the gel degradation.

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“…The abundance of αSMA in the ex vivo cultured tissues indicates MFB contractility. Nevertheless, our model could benefit from incorporation of an isometric force transducer 27,28 . Regarding technical and biological variability, our method is reproducible (viability, ECM network) and less prone to tissue alteration due to short time of culture.…”

Section: Discussionmentioning

confidence: 99%

Human Dupuytren's <em>Ex Vivo</em> Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions

Karkampouna

Kloen

Obdeijn

et al. 2015

JoVE

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Citation: Karkampouna, S., Kloen, P., Obdeijn, M.C., Riester, S.M., van Wijnen, A.J., Kruithof-de Julio, M. Human Dupuytren's Ex Vivo Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions. J. Vis. Exp. (98), e52534, doi:10.3791/52534 (2015). AbstractOrgan fibrosis or "scarring" is known to account for a high death toll due to the extensive amount of disorders and organs affected (from cirrhosis to cardiovascular diseases). There is no effective treatment and the in vitro tools available do not mimic the in vivo situation rendering the progress of the out of control wound healing process still enigmatic.To date, 2D and 3D cultures of fibroblasts derived from DD patients are the main experimental models available. Primary cell cultures have many limitations; the fibroblasts derived from DD are altered by the culture conditions, lack cellular context and interactions, which are crucial for the development of fibrosis and weakly represent the derived tissue. Real-time PCR analysis of fibroblasts derived from control and DD samples show that little difference is detectable. 3D cultures of fibroblasts include addition of extracellular matrix that alters the native conditions of these cells.As a way to characterize the fibrotic, proliferative properties of these resection specimens we have developed a 3D culture system, using intact human resections of the nodule part of the cord. The system is based on transwell plates with an attached nitrocellulose membrane that allows contact of the tissue with the medium but not with the plastic, thus, preventing the alteration of the tissue. No collagen gel or other extracellular matrix protein substrate is required. The tissue resection specimens maintain their viability and proliferative properties for 7 days. This is the first "organ" culture system that allows human resection specimens from DD patients to be grown ex vivo and functionally tested, recapitulating the in vivo situation. Video LinkThe video component of this article can be found at

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A culture force monitor for measurement of contraction forces generated in human dermal fibroblast cultures: evidence for cell-matrix mechanical signalling

Cited by 187 publications

References 14 publications

Endothelin-1 Promotes Myofibroblast Induction through the ETA Receptor via a rac/Phosphoinositide 3-Kinase/Akt-dependent Pathway and Is Essential for the Enhanced Contractile Phenotype of Fibrotic Fibroblasts

Endothelin-1 Promotes Myofibroblast Induction through the ETA Receptor via a rac/Phosphoinositide 3-Kinase/Akt-dependent Pathway and Is Essential for the Enhanced Contractile Phenotype of Fibrotic Fibroblasts

A novel collagen gel-based measurement technique for quantitation of cell contraction force

Human Dupuytren's <em>Ex Vivo</em> Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions

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