Measurement of mechanical tractions exerted by cells in three-dimensional matrices

Abstract

Quantitative measurements of cell-generated forces have heretofore required that cells be cultured on two-dimensional substrates. We describe a technique to quantitatively measure threedimensional traction forces exerted by cells fully encapsulated within well-defined elastic hydrogel matrices. We apply this approach to measure tractions from a variety of cell types and contexts, and reveal patterns of force generation attributable to morphologically distinct regions of cells as they extend into the surrounding matrix.Cells are constantly probing, pushing and pulling on the surrounding extracellular matrix (ECM). These cell-generated forces drive cell migration and tissue morphogenesis and maintain the intrinsic mechanical tone of tissues 1, 2. Such forces not only guide mechanical and structural events, but also trigger signaling pathways that promote functions ranging from proliferation to stem cell differentiation3 , 4. Therefore, precise measurements of the spatial and temporal nature of these forces are essential to understanding when and where mechanical events come to play in both physiological and pathological settings.Methods employing planar elastic surfaces or arrays of flexible cantilevers have mapped, with subcellular resolution, the forces that cells generate against their substrates1 , 5 -7 . However, many processes are altered when cells are removed from native three-dimensional (3D) environments and cultured on two-dimensional (2D) substrates. At a structural level, cells encapsulated within a 3D matrix exhibit dramatically different morphology, cytoskeletal organization, and focal adhesion structure from those on 2D substrates 8 . Even the initial means by which cells attach and spread against a 2D substrate are quite different from the invasive process required for cells to extend inside a 3D matrix. These differences suggest that dimensionality alone may significantly impact how cellular forces are generated and transduced into biochemical or structural changes. Yet, although the mechanical properties of 3D ECMs and the cellular forces generated therein have been shown to * Correspondence should be addressed to C.S. Chen, [C.S. Chen (chrischen@seas.upenn.edu, Tel: 01-215-746-1750, Fax: 01-215-746-1752 COMPETING INTERESTS STATEMENTThe authors declare no competing financial interests. Here, we quantitatively measure the traction stresses (force per area), hereafter tractions, exerted by cells embedded within a hydrogel matrix. GFP-expressing fibroblasts were encapsulated within mechanically well-defined polyethylene glycol (PEG) hydrogels that incorporate proteolytically degradable domains in the polymer backbone and pendant adhesive ligands 10 . The incorporation of adhesive and degradable domains permits the cells to invade, spread, and adopt physiologically relevant morphologies ( Fig. 1a and Supplementary Movie 1). The hydrogels used in this study had a Young's modulus of 600 to 1,000 Pa ( Supplementary Fig. 1), a range similar to commonly used ECMs such as reconstituted collagen ...

Magnetically actuated cell-laden microscale hydrogels for probing strain-induced cell responses in three dimensions

“…[10][11][12] Thus, there is a pressing need for tools to define the mechanical responses of cells in 3D cultures, and a number of coupled experimental and analytical systems have been developed for this purpose. [13][14][15] These mechanobiological tools include ring-like tissue constructs, 16 tissue constructs adhered to flexible substrata 17 and 3D traction microscopy. 14 However, challenges persist for achieving displacement control, obtaining statistically significant sample sizes, avoiding diffusion barriers for soluble factors and avoiding perturbations introduced by sample handling before mechanical loading.…”

“…[13][14][15] These mechanobiological tools include ring-like tissue constructs, 16 tissue constructs adhered to flexible substrata 17 and 3D traction microscopy. 14 However, challenges persist for achieving displacement control, obtaining statistically significant sample sizes, avoiding diffusion barriers for soluble factors and avoiding perturbations introduced by sample handling before mechanical loading.…”

Measuring dynamic cell–material interactions and remodeling during 3D human mesenchymal stem cell migration in hydrogels

“…Although 2D studies add to our understanding of cell-matrix interactions, 2D environments can unnaturally polarize cells, and some aspects of cell motility can be quite different in 2D versus 3D environments (21,22). For these reasons, recent developments have focused on strategies to measure cell-material interactions in three dimensions (e.g., cell-laden hydrogels).…”

The microscope makers

“…He embedded fluorescent beads in the material surrounding a growing mammalian cell so that as the cell moved, it would deform the material, moving the beads. By measuring how much the beads moved, Legant could calculate the forces exerted by the cell 1 . Still, he had difficulty getting accurate data.…”