Integrated Biosystems and Biomechanics Laboratory, University of Michigan, Ann Arbor, MI 48109, USA.
Integr Biol (Camb). 2012 Oct;4(10):1289-98. doi: 10.1039/c2ib20134h.
Forces are increasingly recognized as major regulators of cell structure and function, and the mechanical properties of cells, such as cell stiffness, are essential to the mechanisms by which cells sense forces, transmit them to the cell interior or to other cells, and transduce them into chemical signals that impact a spectrum of cellular responses. Here we reported a new whole-cell cell stiffness measurement technique with a subcellular spatial resolution. This technique was based on a novel cell stretching device that allowed for quantitative control and real-time measurements of mechanical stimuli and cellular biomechanical responses. Our strategy involved a microfabricated array of silicone elastomeric microposts integrated onto a stretchable elastomeric membrane. Using a computer-controlled vacuum, this micropost array membrane (mPAM) was activated to apply equibiaxial cell stretching forces to adherent cells attached on the tops of the microposts. The micropost top positions before and after mPAM stretches were recorded using fluorescence microscopy and further utilized to quantify local cell stretching forces and cell area increments. A robust computation scheme was developed and implemented for subcellular quantifications of cell stiffness using the data of local cell stretching forces and cell area increments generated from mPAM cell stretch assays. Our cell stiffness studies using the mPAM revealed strong positive correlations among cell stiffness, cellular traction force, and cell spread area, and illustrated the important functional roles of actin polymerization and myosin II-mediated cytoskeleton contractility in regulating cell stiffness. Collectively, our work reported a new approach for whole-cell stiffness measurements with a subcellular spatial resolution, which would help likely explain the complex biomechanical functions and force-sensing mechanisms of cells and design better materials for cell and tissue engineering and other applications in vivo.
力被越来越多地认为是细胞结构和功能的主要调节剂，细胞的机械特性，如细胞硬度，对于细胞感知力、将力传递到细胞内部或传递到其他细胞以及将其转化为影响细胞一系列反应的化学信号的机制至关重要。在这里，我们报道了一种具有亚细胞空间分辨率的新的全细胞细胞硬度测量技术。该技术基于一种新颖的细胞拉伸装置，允许对机械刺激和细胞生物力学响应进行定量控制和实时测量。我们的策略涉及到一种硅酮弹性体微柱阵列集成在可拉伸弹性体膜上的微制造阵列。使用计算机控制的真空，这种微柱阵列膜（mPAM）被激活，以向附着在微柱顶部的贴壁细胞施加等双轴细胞拉伸力。在 mPAM 拉伸前后使用荧光显微镜记录微柱顶部的位置，并进一步用于定量局部细胞拉伸力和细胞面积增量。开发并实施了一种强大的计算方案，用于使用从 mPAM 细胞拉伸测定中生成的局部细胞拉伸力和细胞面积增量数据对细胞硬度进行亚细胞量化。我们使用 mPAM 进行的细胞硬度研究表明，细胞硬度、细胞牵引力和细胞扩展面积之间存在很强的正相关，并且说明了肌动蛋白聚合和肌球蛋白 II 介导的细胞骨架收缩在调节细胞硬度方面的重要功能作用。总的来说，我们的工作报道了一种具有亚细胞空间分辨率的全细胞硬度测量的新方法，这可能有助于解释细胞的复杂生物力学功能和力感机制，并设计更好的用于细胞和组织工程以及体内其他应用的材料。
