Mechanical characterisation of mammalian cells during monolayer formation using atomic force microscopy
Barkhuisen, Jessica Lee
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Exploring the elasticity of mammalian cells - through interpretation of the Young’s elastic modulus [E] - has become one of the most useful benchmark biomarkers for investigating the relationship between the physiological and mechanical properties of cells. Such investigations have, until now, primarily focussed on interpreting how cells physically respond at the single cell and monolayer developmental stages. Less consideration into how single cell mechanics translates at intermediary and/or higher cell density developmental stages has been fully addressed. Therefore, attempting to investigate and interpret the mechanical properties of a cell at various stages of development e.g. increasing cell numbers and degree of monolayer confluency, will provide further insight into how tissue structures develop, as well as how cell-associated diseases such as cancer progress. Various techniques are utilised when attempting to determine the Young’s modulus of biological samples. One such method, atomic force microscopy (AFM), has emerged as a useful tool for determining the Young’s modulus of soft biological samples. When utilising AFM for the mechanical characterisation of cells, cell samples are often prepared as adherent cultures with a variety of AFM cantilever geometries and experimental parameters utilised. Variation in the selection of AFM parameters between studies can result in variation between tested batches of cell samples. Markedly, when attempting to measure the Young’s modulus of mammalian cell lines by AFM, little consideration has been given regarding how the specific stage of cell development and degree of monolayer confluency can affect derived elasticity outputs. Recent studies have highlighted the apparent differences in the elasticity of cells present at alternate stages of development; single isolated cells present with a higher Young’s modulus compared to cells within a structured cell monolayer. Further to this, AFM indentation and resulting [E] has been reported to be sensitive to the indentation depth analysed. Variation in the selection of an appropriate AFM loading force, and how this can affect the outcome of cell mechanical outputs, such as resulting final cell deformation and derived cell [E] has been not fully considered. Therefore, the primary aim of this thesis was to carry out a defined and bridged approach for investigating cell mechanics at precisely defined stages of monolayer formation. Using an incremental loading force range, together with cell [E] map and morphological fluorescence cell imaging, this thesis demonstrates the variation in derived mammalian cell [E] outputs through different stages of cell monolayer development. With variation in mechanical outputs described and associated to the most relevant intercellular and intracellular cytoskeletal and cell organelle components.