Collective Cell Behavior in Mechanosensing of Substrate Thickness

Abstract

Extracellular matrix stiffness has a profound effect on the behavior of many cell types. Adherent cells apply contractile forces to the material on which they adhere and sense the resistance of the material to deformation-its stiffness. This is dependent on both the elastic modulus and the thickness of the material, with the corollary that single cells are able to sense underlying stiff materials through soft hydrogel materials at low (<10 μm) thicknesses. Here, we hypothesized that cohesive colonies of cells exert more force and create more hydrogel deformation than single cells, therefore enabling them to mechanosense more deeply into underlying materials than single cells. To test this, we modulated the thickness of soft (1 kPa) elastic extracellular-matrix-functionalized polyacrylamide hydrogels adhered to glass substrates and allowed colonies of MG63 cells to form on their surfaces. Cell morphology and deformations of fluorescent fiducial-marker-labeled hydrogels were quantified by time-lapse fluorescence microscopy imaging. Single-cell spreading increased with respect to decreasing hydrogel thickness, with data fitting to an exponential model with half-maximal response at a thickness of 3.2 μm. By quantifying cell area within colonies of defined area, we similarly found that colony-cell spreading increased with decreasing hydrogel thickness but with a greater half-maximal response at 54 μm. Depth-sensing was dependent on Rho-associated protein kinase-mediated cellular contractility. Surface hydrogel deformations were significantly greater on thick hydrogels compared to thin hydrogels. In addition, deformations extended greater distances from the periphery of colonies on thick hydrogels compared to thin hydrogels. Our data suggest that by acting collectively, cells mechanosense rigid materials beneath elastic hydrogels at greater depths than individual cells. This raises the possibility that the collective action of cells in colonies or sheets may allow cells to sense structures of differing material properties at comparatively large distances.

Effect of Different Crosslinking Strategies on Physical Properties and Biocompatibility of Freestanding Multilayer Films Made of Alginate and Chitosan

“…The crosslinking method did not seem to make any difference in this case, and there was no obvious difference compared to the untreated films as well. When a cell adheres to a substrate it responds to the substrate stiffness through the rearrangement of the cytoskeletal components such as actin filament and microtubules . Cells have more organized cytoskeletons on stiffer substrates .…”

Culture on Tissue‐Specific Coatings Derived from α‐Amylase‐Digested Decellularized Adipose Tissue Enhances the Proliferation and Adipogenic Differentiation of Human Adipose‐Derived Stromal Cells

“…ASCs cultured on TCP are expected to have a spindle‐shaped morphology, with cell spreading and flattening serving as markers of senescence in long‐term culture . In general, cell spreading varies in 2D and 3D systems, and correlates with properties such as substrate stiffness, ligand density, and arrangement through the regulation of focal adhesions . As such, the spindle‐shaped morphology observed on the α‐amylase‐digested coatings may be related to their lower stiffness and more fibrous 3D ultrastructure .…”

“…In general, cell spreading varies in 2D and 3D systems, and correlates with properties such as substrate stiffness, ligand density, and arrangement through the regulation of focal adhesions . As such, the spindle‐shaped morphology observed on the α‐amylase‐digested coatings may be related to their lower stiffness and more fibrous 3D ultrastructure . Fibrous topographies have been postulated to provide cells with a greater local surface area with multiple physical sites for engraftment in all three dimensions, favorable for cell attachment, migration, and proliferation .…”

Asamataxis: A cooperative relayed migration in response to subsurface inhomogeneity leads to long-range self-patterning of cells

“…al. showed that the cell colonies can sense the underlying rigid material through a 200 µm thick soft gel, the thickness through which a single cell cannot sense the underlying rigid material 25 . However, while they used cell colonies, we showed that single cells in close vicinity to each other can sense the embedded rigid structure from far away distance due to relay of the strain field via collective traction.…”

“…As the stiffness of the PAA gel increases the zone of deformation of PAA gel due to force applied by cells decreases 26,27 . Also, the depth sensing ability of collective cell is less than 200 µm 25 . Therefore, when the stiffness of PAA gel or top layer of PAA gel was increased, the patterning disappeared.…”