Assembly and Use of a Microfluidic Device to Study Cell Migration in Confined Environments

Cells migrating in tissues must often pass through physical barriers in their surroundings in the form of fibrous extracellular matrix or other cells. To improve our understanding of how cells move in such confined microenvironments, we have designed a microfluidic device in which cells migrate through a series of three-dimensional polydimethylsiloxane (PDMS) constrictions with precisely controlled geometries that mimic physiological pore sizes. The migration device offers an experimental platform that combines a well-defined three-dimensional (3D) environment with a setup well suited for imaging confined cell migration at high spatial and temporal resolution. In this protocol, we describe the fabrication and use of these devices using standard soft lithography techniques and light microscopy. Analysis of live-cell time-lapse series of cells with fluorescently labeled nuclear and/or cytoskeletal structures migrating in the devices can reveal new insights into the molecular processes required for confined migration, including the role of the linker of nucleoskeleton and cytoskeleton (LINC) complex, which has been implicated in 3D migration.