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Dhananjay Tambe, Ramin Zareian, Dimitrios Karamichos, James D. Zieske, Jeffrey W. Ruberti; Continuous Measurement Of Physical Forces During Human Primary Corneal Fibroblast Migration. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1528.
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: Little is known about the mechanobiology of matrix production by corneal fibroblasts which have been shown to self organize and produce extracellular matrix in vitro. During coordinated migration, epithelial and endothelial cells apply forces to the substrate as well as to their neighbors [Trepat et al 2009 Tambe et al 2011]. If such forces are generated by fibroblasts during matrix production, they may influence the deposition and retention of collagenous matrix [Zareian et al 2010]. However it is very difficult to follow fibroblast behavior while measuring forces on time scales long enough to permit matrix production. Here we present an in vitro mechanobiological assay capable of directly observing the behavior of PHCF over relatively long time scales while simultaneously measuring intrinsic (cell-cell and cell-substrate) physical forces.
We combine the mechanobioreactor described in [Paten et al 2011] with a 100um thick, collagen coated, calibrated polyacrylamide gel [E=1250 Pa Yeung et al 2005] to observe the mechanobiology of PHCF culture. Forces applied to the substrate are interrogated continuously via embedded 0.5um fluorescent beads. Using Fourier transform traction microscopy the gel deformation is transformed into a continuous traction vector field applied by the cells to the substrate. Using monolayer stress microscopy the traction field is transformed into stresses applied by the cells to their neighbors.
Over the time course of this experiment, the corneal fibroblasts actively migrate over the gel surface and begin to form a confluent monolayer. We observe that cells gradually build up tractions. When they self organize, the traction at a point has contributions from two sources, contraction generated by cells attached at that location, and contractions generated by cells far from that location but transmitted through cell-cell contacts. Magnitude of this traction is on the order of 150Pa.
The combination of the mechanobioreactor with the traction force microscopy method permits direct measurement PHCF physical forces during migration and growth to confluence. The system will ultimately enable dynamic, continuous and quantitative observation of the mechanobiology of matrix production.
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