CONFINED COMPRESSION OF SINGLE CELLS USING AFM

描述（由申请人提供）：美国死亡和残疾的两大主要原因是心血管疾病和骨关节炎，每年导致近1400亿美元的相关医疗费用。在我们理解疾病和修复的细胞过程中的一个主要障碍是对组成细胞的内在材料特性的有限了解，这些特性影响细胞的形状、变形能力、运动性、分裂、活力和ECM的组织。单细胞的中尺度生物力学研究对于破译宏观尺度器官和组织水平的力如何传递到细胞内的纳米尺度分子机制至关重要。对于给定的细胞类型，粘弹性材料常数的报告值可以在几个数量级上变化，这表明标准的测试方法尚未出现，并且与体内环境相关的固有细胞特性尚未得到充分鉴定。到目前为止，很少有研究直接解释细胞固有的固体-流体组成，也没有研究使用完全限制的实验测试方法来简化系统并提取感兴趣的细胞特性。本申请的具体目的是：目的1：开发第一个单细胞限制压缩装置，使用定制的原子力显微镜（AFM）和微加工的测试室，分析基于多相混合物理论。目标二：探索单细胞内双相材料特性的细胞内深度依赖性，使用AFM和一种新的侧视有限压缩室，实时共聚焦显微镜的活细胞和数字图像相关性定量区域细胞内变形。这种异质性水平在1-2 μ m的范围内，介于整个细胞和单个细胞骨架丝和蛋白质之间。这将使我们能够测试的假设，区域变化的细胞内结构引起不均匀的细胞内变形，以响应均匀施加的负载。这项探索性研究的成功结果将有助于关闭细胞机械传导机制的多尺度研究，激发新的研究问题，并导致更好地理解和治疗承重组织的病理学（如心血管疾病和骨关节炎），其中生物力学因素起着重要作用。

DESCRIPTION (provided by applicant): The two leading causes of death and disability in the U.S. are cardiovascular disease and osteoarthritis, which result in nearly $140 billion per year in related healthcare costs combined. A major hurdle in our understanding of the cellular processes underlying disease and repair in such load-bearing tissues is a limited knowledge of the intrinsic material properties of the constituent cells, which impact cell shape, deformability, motility, division, viability, and organization of the ECM. Studies of the meso-scale biomechanics of single cells are critical for deciphering how macro-scale organ and tissue-level forces are transmitted to the nano-scale molecular machinery within the cell. Reported values of viscoelastic material constants for a given cell type can vary over several orders of magnitude, suggesting that a standard testing method has not yet emerged, and the intrinsic cell properties pertinent to the in vivo environment have yet to be adequately identified. Few studies to date have directly accounted for the inherent solid-fluid composition of the cell and no studies have used a fully-confined experimental testing methodology to simplify the system and extract the cellular properties of interest. The specific aims of this application are: Aim 1: To develop the first single-cell confined compression apparatus using customized atomic force microscopy (AFM) and a microfabricated test chamber, with analysis based on multiphasic mixture theory. Aim 2: To explore the intracellular depth-dependence of biphasic material properties within single cells using AFM and a novel side-view confined compression chamber with real-time confocal microscopy of living cells and digital image correlation for quantifying regional intracellular deformation. This level of heterogeneity is on a scale of 1-2 fm that is intermediate between the whole cell and individual cytoskeletal filaments and proteins. This will allow us to test the hypothesis that regional variations in intracellular structure give rise to nonuniform intracellular deformations in response to a uniformly applied load. Successful outcomes of this exploratory research would help close the loop on the multi-scale investigation of cellular mechanotransduction mechanisms, motivating new research questions and leading to an improved understanding and treatment of pathologies of load-bearing tissues (such as cardiovascular disease and osteoarthritis) in which biomechanical factors play a significant role.