Cell motility regulation on a stepped micro pillar array device (SMPAD) with a discrete stiffness gradient

Our tissues consist of individual cells that respond to the elasticity of their environment, which varies between and within tissues. To better understand mechanically driven cell migration, it is necessary to manipulate the stiffness gradient across a substrate. Here, we have demonstrated a new variant of the microfabricated polymeric pillar array platform that can decouple the stiffness gradient from the ECM protein area. This goal is achieved via a "stepped'' micro pillar array device (SMPAD) in which the contact area with the cell was kept constant while the diameter of the pillar bodies was altered to attain the proper mechanical stiffness. Using double-step SU-8 mold fabrication, the diameter of the top of every pillar was kept uniform, whereas that of the bottom was changed, to achieve the desired substrate rigidity. Fibronectin was immobilized on the pillar tops, providing a focal adhesion site for cells. C2C12, HeLa and NIH3T3 cells were cultured on the SMPAD, and the motion of the cells was observed by time-lapse microscopy. Using this simple platform, which produces a purely physical stimulus, we observed that various types of cell behavior are affected by the mechanical stimulus of the environment. We also demonstrated directed cell migration guided by a discrete rigidity gradient by varying stiffness. Interestingly, cell velocity was highest at the highest stiffness. Our approach enables the regulation of the mechanical properties of the polymeric pillar array device and eliminates the effects of the size of the contact area. This technique is a unique tool for studying cellular motion and behavior relative to various stiffness gradients in the environment.