title: Stiffness-controlled three-dimensional extracellular matrices for high-resolution imaging of cell behavior.
abstract: Regulation of cell functions by the physical properties of the extracellular matrix (ECM) has emerged as a crucial contributor to development and disease. Two specific physical properties of the ECM, stiffness and dimensionality, each influence cell signaling and function. As these ECM physical properties are linked to other properties that also regulate cell behavior, e.g., integrin ligand density, parsing the specific contributions of ECM stiffness and dimensionality has proven difficult. Here we detail a simple protocol, which can be completed in 1-2 d, for combining three-dimensional (3D) ECM engagement with controlled underlying ECM stiffness. In these 'sandwich gels', cells are sandwiched between a 3D fibrillar ECM and an ECM-coupled polyacrylamide gel of defined compliance, allowing the study of the specific effects of ECM compliance on cell function in physiologically relevant 3D ECMs. This type of system enables high-resolution time-lapse imaging and is suitable for a wide range of cell types and molecular perturbations.
authors.affiliation: Cell Biology and Physiology Center, National Heart, Lung, and Blood Institute (NHBLI), US National Institutes of Health (NIH), Bethesda, Maryland, USA. fischerr2@nhlbi.nih.gov
authors.forename: Robert S
authors.initials: RS
authors.lastname: Fischer
authors.forename: Kenneth A
authors.initials: KA
authors.lastname: Myers
authors.forename: Margaret L
authors.initials: ML
authors.lastname: Gardel
authors.forename: Clare M
authors.initials: CM
authors.lastname: Waterman
mesh_terms: D000180:Acrylic Resins; D002450:Cell Communication; D018929:Cell Culture Techniques; D060833:Cellular Microenvironment; D005109:Extracellular Matrix / Q000737:chemistry* / Q000648:ultrastructure; D061307:Human Umbilical Vein Endothelial Cells; D008858:Microscopy, Phase-Contrast
chemical_list: D000180:Acrylic Resins; C016680:polyacrylamide gels