Cellular mechanosensing on a cell-scale stiffness gradient substrate

Cells have the ability to sense and respond to various mechanical cues from their immediate surroundings. One of the manifestations of such a process, which is also known as "mechanosensing", is directed cell migration. Various biological processes have been shown to be controlled by extracellular matrix (ECM) stiffness. Substrates with a high stiffness gradient have been used as a platform to investigate cellular motion in response to mechanical cues. However, creating a cell scale stiffness gradient in such a cell adhesion friendly substrate still remains elusive. In this study, we present a simple and versatile method for fabricating substrates with a periodically varying stiffness profile at the cellular scale, featuring customizable high stiffness gradients. Fibroblast cells, when presented with such continuous yet anisotropic variation of stiffness, preferentially position their nuclei in stiffer regions of the substrate and align themselves along the direction of the lowest rigidity gradient. Furthermore, when the rigidity of the substrate is sufficiently high, cells exhibit less sensitivity to stiffness gradients, with their elongation and nuclear positioning becoming independent of stiffness variations. Overall, our experimental results reveal new insights into the process of cellular mechanosensing where the cell-scale gradient drives strong positional and orientational order.