Cell Biology and Physiology Center, National Heart, Lung, and Blood Institute (NHBLI), US National Institutes of Health (NIH), Bethesda, Maryland, USA.
Nat Protoc. 2012 Nov;7(11):2056-66. doi: 10.1038/nprot.2012.127. Epub 2012 Oct 25.
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.
细胞功能受细胞外基质（ECM）物理特性的调节，这已成为发育和疾病的一个关键因素。ECM 的两个特定物理特性，即刚度和维度，都会影响细胞信号转导和功能。由于这些 ECM 物理特性与其他也能调节细胞行为的特性（例如整合素配体密度）相关联，因此解析 ECM 刚度和维度的具体贡献一直很困难。在这里，我们详细介绍了一种简单的方案，该方案可以在 1-2 天内完成，将三维（3D）ECM 结合与受控的基础 ECM 刚度结合在一起。在这些“夹层凝胶”中，细胞被夹在 3D 纤维 ECM 和具有确定顺应性的 ECM 偶联聚丙烯酰胺凝胶之间，从而可以研究 ECM 顺应性对细胞在生理相关 3D ECM 中功能的具体影响。这种类型的系统支持高分辨率延时成像，适用于多种细胞类型和分子扰动。
