CONFINED COMPRESSION OF SINGLE CELLS USING AFM

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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.