Generation ofOrthogonal Gradients of the Matrix Stiffnessand Chemotactic Cues in a Suspended Array of Hydrogel to Study hMSCs Migration

Stem cell migration is a tightly regulated process in vivo, orchestrated by a collection of mechanical and chemotactic cues via concentration gradients. A variety of in vitro assays have been developed to facilitate cell migration studies; however, very few assays allow the investigation of both matrix stiffness and chemotactic cues on cell migration within a single device, especially in a three-dimensional (3D) environment. Here, we develop a microfluidic device that can produce 3D orthogonal gradients of matrix stiffness and chemotactic cues with varied steepness in a suspended array of hydrogel cylinders. The device’s working principle is the formation of diffusion-driven concentration gradients within a suspended array of hydrogel cylinders between a source and a sink. Device fabrication is based on poly(dimethylsiloxane) (PDMS) replica molding, followed by assembly on a glass substrate. To validate this device, we study the migration of human mesenchymal stem cells (hMSCs) in response to orthogonal gradients of matrix stiffness and stromal cell-derived factor 1 alpha (SDF-1α). This technology has the potential to be applied to various cell types, facilitating exploration in different cellular contexts.