Collective cell behaviour in long-range mechanosensing of extracellular matrix dimensions

Tissue healing and regeneration is strongly influenced by the mechanical properties of the extracellular matrix (ECM). Most mammalian cells attach to ECM and, by applying force, are able to mechanosense its stiffness as a result of its resistance to deformation. An increase in stiffness is known to affect cell proliferation, differentiation and migration. However, the stiffness that cells detect is determined not only by the matrix elastic modulus but also by the material thickness. Single cells are known to sense rigid boundaries through a soft hydrogel when the depth is less than 10 μm. However, most of the tissues are composed of cohesive layers of cells, which behave very differently from single cells. Therefore, this study tested the hypothesis that colonies exert more force by acting collectively and produce more deformation than individual cells, allowing them to mechanosense more deeply into the underlying substrate than individual cells.The effect of substrate elasticity and thickness on cells and colonies was determined by culturing cells on customised extracellular-matrix-coated polyacrylamide (PA) hydrogels of varying elasticity (0.5 – 40 kPa) and thickness (1 – 1000 μm), attached basally to glass substrates. Cell spreading, attachment and density were measured by epifluorescence and confocal microscopy. Cell-induced surface deformations were quantified by imaging the fluorescent fiducial-marker-labelled hydrogels during a time-lapse experiment and by computing cumulative deformations using MATLAB code. Immunofluorescent of E-cadherin and involucrin was used to test phenotypical changes in primary keratinocytes.MG63 (human osteosarcoma) single cells area decreased as a function of substrate thickness; the data was fitted to an exponential model, characterised by a half-maximum response at 3.2 μm thickness. MG63 colonies were found to mechanosense the stiffness of the underlying support at greater thicknesses than individual cells. The half-maximal response for MG63 colonies (54 µm) was 17 times greater than that for individual cells. The abilityof cells to mechanosense matrix depth was dependent on Rho-associated protein kinase mediated cellular contractility. Colony-induced surface deformations were significantly greater on thick hydrogels than on thin hydrogels (3.9 ± 0.8 versus 1.9 ± 1.2 μm; p < 0.05). In addition, deformations extended greater distances from the colony border on thick hydrogels compared to thin hydrogels. A fibrous ECM protein (Col) was found to amplify the depth at which both single cells and colonies sense the underlying rigid substrate. The expression of E-cadherin in keratinocytes colonies was identified only on thin hydrogels. Overall, the novel findings presented in this thesis suggest that by acting collectively, groups of cells mechanosense stiff boundaries at greater thicknesses than single cells. This integrative phenomenon may empower colonies or sheets of cells to interrogate their environment mechanically in a range of biological contexts such as tissue regeneration, embryogenesis, cancer metastasis or biomaterials design

Poly(N-isopropylacrylamide) based thin microgel films for use in cell culture applications

Poly(N-isopropylacrylamide) (PNIPAm) is widely used to fabricate cell sheet surfaces for cell culturing, however copolymer and interpenetrated polymer networks based on PNIPAm have been rarely explored in the context of tissue engineering. Many complex and expensive techniques have been employed to produce PNIPAm-based films for cell culturing. Among them, spin coating has demonstrated to be a rapid fabrication process of thin layers with high reproducibility and uniformity. In this study, we introduce an innovative approach to produce anchored smart thin films both thermo- and electro-responsive, with the aim to integrate them in electronic devices and better control or mimic different environments for cells in vitro. Thin films were obtained by spin coating of colloidal solutions made by PNIPAm and PAAc nanogels. Anchoring the films to the substrates was obtained through heat treatment in the presence of dithiol molecules. From analyses carried out with AFM and XPS, the final samples exhibited a flat morphology and high stability to water washing. Viability tests with cells were finally carried out to demonstrate that this approach may represent a promising route to integrate those hydrogels films in electronic platforms for cell culture applications

Collective cell behavior in mechanosensing of substrate thickness

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 (&lt;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.</p