A 4D Traction Force Microscope for the mapping of cellular mechanical stresses

描述（由申请人提供）：细胞产生的机械应力在许多生理过程中是至关重要的，包括胚胎发生，细胞迁移，细胞增殖和组织形成。然而，目前还没有方法可以测量三维支架在粘附和迁移过程中施加的细胞牵引力。这一信息对于理解细胞-细胞和细胞-细胞外基质（ECM）相互作用在伤口愈合、癌症转移、心血管疾病和慢性炎症等过程中的作用至关重要。为了部分解决这一需求，几年前开发了一种称为牵引力显微镜的技术，用于量化和绘制细胞在其基底上产生的牵引场（1,2）。这项技术提供了关于细胞迁移和粘附机制的宝贵信息，并证明了正常和患病状态机制的关键差异。然而，这种目前使用的技术是有限的——最先进的技术是平面基底上细胞产生的力的二维计算。众所周知，2D基质缺乏3D ECM在体内为细胞提供的生理现实环境。因此，为了更准确地再现细胞微环境，这些测量需要在3-D中进行，其中细胞的所有侧面都能够与细胞外基质结合，使基质变形，并利用细胞产生的牵引应力进行迁移。该提案旨在开发和构建一种仪器（硬件）和一种计算算法（软件），用于计算嵌入在3D矩阵中的细胞施加的应力，以研究模拟原生体内环境的ecm中细胞粘附和迁移的动态过程。我们期望，就像二维牵引力显微镜已经广泛实施；我们的工具将广泛引起对许多不同生理系统和疾病条件下细胞迁移和粘附机制感兴趣的大量科学家的兴趣。

DESCRIPTION (provided by applicant): Cell-generated mechanical stresses are critical during a number of physiological processes, including embryogenesis, cell migration, cell proliferation and tissue formation. However, there is no method currently available to measure cellular traction forces exerted during adhesion and migration in 3-D scaffolds. This information is critical for understanding the contribution of cell-cell and cell-extracellular matrix (ECM) interactions in processes such as wound healing, cancer metastasis, cardiovascular disease, and chronic inflammation. To partially address this need, a technique called Traction Force Microscopy was developed several years ago to quantify and map the traction field created by a cell on its substrate (1, 2). This technique has provided valuable information about the mechanisms of cell migration and adhesion and has demonstrated key differences in the mechanics of normal and diseased states. However, this currently used technique is limited--- the state-of-the-art is a 2-D calculation of the cell-generated forces on a planar substrate. It is well-established that 2D substrates lack the physiologically realistic environment that a 3D ECM provides cells in vivo. Therefore, to more accurately recapitulate the cellular microenvironment, these measurements need to be done in 3-D, where all sides of the cell are capable of binding to the extracellular matrix, deforming the substrate and using cell-generated traction stresses to migrate. The proposal seeks to develop and build an instrument (hardware) and a computational algorithm (software) that calculates the stresses exerted by cells embedded in 3D matrices for studies of the dynamic processes of cell adhesion and migration in ECMs which mimic the native in vivo environment. We expect that just as 2D Traction Force Microscopy has been widely implemented; our tool will be of wide-spread interest to the large community of scientists interested in mechanism of cell migration and adhesion for a number of different physiological systems and disease conditions. PUBLIC HEALTH RELEVANCE (provided by applicant): In this proposal, we plan to develop a 4D traction force microscope (hardware and software) that can measure the forces that cells exert against their substrate during adhesion and migration in 3D space and time. Such a device is essential to understand the molecular machinery that drives cell migration in physiological processes such as wound healing, tissue formation, and during the progression of diseases including cancer metastasis and atherosclerosis.