CONFINED COMPRESSION OF SINGLE CELLS USING AFM

描述(申请人提供)：在美国，导致死亡和残疾的两大原因是心血管疾病和骨关节炎，这两种疾病每年导致的相关医疗费用加起来接近1400亿美元。在我们理解这些承载组织中潜在的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属性的有限的了解，这些材料属性影响细胞的形状、变形性、运动性、分裂、活性和ECM的组织。研究单个细胞的中观生物力学对于破译宏观器官和组织水平的力是如何传递到细胞内的纳米级分子结构至关重要。已报道的一种细胞类型的粘弹性材料常数的值可能会在几个数量级上变化，这表明标准的测试方法尚未出现，与体内环境相关的固有细胞属性尚未得到充分识别。到目前为止，很少有研究直接解释细胞固有的固液组成，也没有研究使用完全受限的实验测试方法来简化系统并提取感兴趣的细胞属性。这项应用的具体目标是：目标1：利用定制的原子力显微镜(AFM)和微型制造的试验室，开发第一台基于多相混合物理论分析的单细胞受限加压装置。目的：利用原子力显微镜(AFM)和一种新型的侧视受限加压小室，利用活细胞的实时共聚焦显微镜和数字图像相关技术对局部细胞内的变形进行量化，探讨单细胞内双相材料性质随细胞内深度的变化。这种异质性水平是介于整个细胞和单个细胞骨架细丝和蛋白质之间的1-2 fM。这将使我们能够检验这样的假设，即细胞内结构的区域变化会引起细胞内非均匀变形，以响应均匀施加的载荷。这项探索性研究的成功结果将有助于结束多尺度细胞机械转导机制研究的循环，引发新的研究问题，并导致对生物力学因素起重要作用的承重组织(如心血管疾病和骨关节炎)的病理更好的理解和治疗。

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.