Isolation, Extraction, and Analysis of Cells After Confined Migration

Cell migration through confined microenvironments is a critical biological process that underlies numerous physiological and pathological events, including immune cell trafficking, tissue morphogenesis, and cancer metastasis. Although polydimethylsiloxane-based microchannel devices have enabled detailed studies of confined migration, the efficient collection of cells post-migration for downstream molecular analyses remains a major challenge. Existing approaches often rely on harsh mechanical dissociation that compromises cell viability and integrity and do not permit in situ collection of cell lysates. To overcome these limitations, we have developed the Trap-based Recovery After Permeation (TRAP) chip, a pump-free microfluidic platform that integrates controlled confined migration with efficient post-migration cell or lysate collection. The TRAP chip incorporates microchannel arrays terminating in a precisely engineered trap region that enables gentle recovery of cells or cellular components without exposing them to high shear forces or requiring large buffer volumes. This innovation ensures the viability of recovered cells and expands the applicability of confined migration assays beyond imaging-based studies. We demonstrate that the TRAP chip facilitates the extraction of post-confinement cells for mechanical characterization, including measurement of Young's modulus, as well as the isolation of proteins and RNA suitable for downstream assays such as western blot and qPCR. The TRAP chip thus represents a significant advancement in microfluidic technologies, offering a robust, reproducible, and minimally invasive approach for studying the mechanobiology of confined migration, with broad potential for applications in basic research, cellular engineering, and translational studies where cell behavior under physical confinement is of critical importance. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Fabrication of TRAP and control chips Basic Protocol 2: Cell seeding and live cell isolation from TRAP chips Alternate Protocol: Biomolecular extraction from TRAP chips.