Laboratoire Matière et Systèmes Complexes (MSC), Université Paris-Diderot & CNRS UMR 7057, Bâtiment Condorcet, Paris, France.
Lab Chip. 2011 Mar 7;11(5):805-12. doi: 10.1039/c0lc00221f. Epub 2010 Dec 6.
Most tissue cells evolve in vivo in a three-dimensional (3D) microenvironment including complex topographical patterns. Cells exert contractile forces to adhere and migrate through the extracellular matrix (ECM). Although cell mechanics has been extensively studied on 2D surfaces, there are too few approaches that give access to the traction forces that are exerted in 3D environments. Here, we describe an approach to measure dynamically the contractile forces exerted by fibroblasts while they spread within arrays of large flexible micropillars coated with ECM proteins. Contrary to very dense arrays of microposts, the density of the micropillars has been chosen to promote cell adhesion in between the pillars. Cells progressively impale onto the micropatterned substrate. They first adhere on the top of the pillars without applying any detectable forces. Then, they spread along the pillar sides, spanning between the elastic micropillars and applying large forces on the substrate. Interestingly, the architecture of the actin cytoskeleton and the adhesion complexes vary over time as cells pull on the pillars. In particular, we observed less stress fibers than for cells spread on flat surfaces. However, prominent actin stress fibers are observed at cell edges surrounding the micropillars. They generate increasing contractile forces during cell spreading. Cells treated with blebbistatin, a myosin II inhibitor, relax their internal tension, as observed by the release of pillar deformations. Moreover, cell spreading on pillars coated with ECM proteins only on their tops are not able to generate significant traction forces. Taken together, these findings highlight the dynamic relationship between cellular forces and acto-myosin contractility in 3D environments, the influence of cytoskeletal network mechanics on cell shape, as well as the importance of cell-ECM contact area in the generation of traction forces.
大多数组织细胞在包括复杂拓扑模式的三维（3D）微环境中进行体内进化。细胞通过细胞外基质（ECM）施加收缩力以粘附和迁移。尽管细胞力学已在 2D 表面上得到广泛研究，但很少有方法可以获得在 3D 环境中施加的牵引力。在这里，我们描述了一种方法，可用于测量在涂有 ECM 蛋白的大柔性微柱阵列中扩散的成纤维细胞所施加的收缩力。与非常密集的微柱阵列相反，选择微柱的密度以促进柱之间的细胞粘附。细胞逐渐刺穿微图案化的基底。它们首先在不施加任何可检测力的情况下粘附在柱子的顶部。然后，它们沿着柱子的侧面扩散，跨越弹性微柱并在基底上施加很大的力。有趣的是，随着细胞在柱子上拉动，肌动球蛋白细胞骨架和粘附复合物的结构随时间而变化。特别是，我们观察到的应力纤维比在平坦表面上扩散的细胞少。然而，在围绕微柱的细胞边缘观察到明显的肌动蛋白应力纤维。它们在细胞扩展过程中产生不断增加的收缩力。用肌球蛋白 II 抑制剂 blebbistatin 处理的细胞会释放柱变形，从而松弛其内部张力。此外，仅在顶部涂有 ECM 蛋白的柱子上的细胞无法产生显著的牵引力。综上所述，这些发现强调了 3D 环境中细胞力与肌动球蛋白收缩性之间的动态关系、细胞骨架网络力学对细胞形状的影响，以及细胞-ECM 接触面积在产生牵引力中的重要性。
