Book Detail:
Author: Claire M. Elkins
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ISBN:
Size: 44.40 MB
Format: PDF, ePub, Mobi
Release Date: 2014
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Language: en
View: 4513
Status: Available
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Book Description
Download PDF A Cell Monolayer Rheometer eBook. You can read online on your kindle, Android, iPhone, iPad. Over the past three decades, a growing body of research has shown that the mechanical interaction between cells and their physical environment plays an extensive role in controlling protein expression and cell behavior. Structurally-associated proteins have been implicated in cell-signaling pathways as diverse as cell proliferation, differentiation, inflammation, and apoptosis. Thus, it is becoming increasingly apparent that the structure and mechanical properties of the cell must be better understood in order to fully understand cell behavior. To this end, researchers have developed a variety of methods for measuring the mechanical properties of whole cells, including micropipette aspiration, magnetic particle cytometry, traction force microscopy, AFM, and single-cell tensile testing. Though all of these techniques provide insight into cell mechanics, most also involve some non-ideal conditions for acquiring live cell data. As a result, current techniques can be time-consuming and potentially fail to capture the true behavior of a healthy, confluent monolayer. We have attempted to address the need for more rapid, accurate measurement of cell mechanical properties via the construction of a linear cell monolayer rheometer (LCMR). The LCMR is an apparatus capable of probing the average mechanical properties of an entire monolayer of adherent cells. In standard operation, an adherent monolayer of cells is gently compressed from above by an adhesion protein-coated top plate attached to a piezoelectric stage. After a waiting period, the top plate is sheared laterally, thereby inducing a shearing deformation of the cell layer. A force transducer attached to the top plate collects stress data for each step strain motion. The entire apparatus is mounted on an inverted microscope, allowing live cell imaging throughout the experiment. There are several advantages to the LCMR technique for probing cell mechanical properties. The first is the ability to rapidly obtain average mechanical properties for an entire population of cells, thereby greatly reducing the number of measurements necessary to represent the cell population. Second, cells are tested while maintaining their adherent shape and cell-to-cell contacts. Finally, the LCMR facilitates live cell imaging as the cells are sheared. This feature allows cell-substrate attachments to be visualized continuously and enables accurate calculation of both the contact area and the strain undergone by the cell layer. We have employed the LCMR to conduct investigations into the mechanical behavior of two model systems. The first study involved murine stromal vascular cells systematically inhibited for three major cytoskeletal components using either drugs (to induce either actin or tubulin deficiency) or genetically deficient organisms (for vimentin-deficient cells). Subsequent measurement of the cell relaxation modulus with the LCMR revealed that both actin- and vimentin-deficient cells had ~50% lower relaxation modulus values than wild-type, while tubulin deficiency resulted in ~100% higher relaxation modulus values. These results provide new quantitative evidence for the contrasting roles actin microfilaments, vimentin intermediate filaments, and tubulin microtubules play in determining whole cell mechanics. A second LCMR study examined the mechanical interaction between human corneal epithelial cells and contact lens hydrogel materials. During contact lens wear, components of the human tear film are known to accumulate on the lens, forming a protein-rich conditioning film. The effect of this conditioning film on bacterial adhesion is well-known; however, the effect of such protein deposition on lens interactions with the corneal epithelium remains largely unexplored. To investigate this effect we utilized the LCMR to quantify human corneal epithelial cell adhesion to soft contact lenses that were fouled with the tear film protein lysozyme. For both lens types tested, the presence of lysozyme increased corneal cell adhesion to the contact lens, with the apparent relaxation modulus increasing up to an order of magnitude in the presence of the protein. The magnitude of this increase depended on the type of buffer solution: lenses soaked in borate-buffered solutions exhibited a much greater increase in cell attachment upon protein addition than those soaked in phosphate-buffered solutions. These findings highlight a potentially deleterious consequence of protein fouling on lens interactions with the human cornea, and suggest lens soaking solutions could perhaps be tuned to mitigate this effect.
