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Jeffrey Ruberti, Ramin Zareian, Dimitrios Karamichos, Dhananjay Tambe, Monica Susilo, Jeffrey Paten, Nima Saeidi, James D Zieske; Long-term, Live-Imaging of the Mechanodynamics of Primary Human Corneal Fibroblasts: From Initial Seeding to Matrix Production. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):1934.
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© ARVO (1962-2015); The Authors (2016-present)
In this investigation, we asked whether a small mechanical force or "bias" applied to a natural substrate like collagen, could fundamentally alter the patterning behavior and the dynamics of cell organization and the organization of the matrix secreted by the cell population. Our further goal was to extract numerous quantitative measures of the influence of applied load on the emergent behavior of cell colony systems.
Long-term 4-D microscopy, enabled by a custom-designed, optically-accessible mechano-bioreactor, was used to examine a primary human corneal fibroblast (PHCF) colony cultured on three different substrates: a mechanically-loaded dense, disorganized collagen substrate (DDCS), a glass coverslip and an unloaded DDCS. The imaging comprised both differential interference contrast (DIC) and fluorescence microscopy (FM). In the case of DIC, images were taken at 20x at three locations per substrate, once every six minutes for up to twelve days. For the FM, images were taken every hour. In total, we have accumulated more than 7000 hours of video data. To quantify the alignment and migration behavior of cell colony, we applied particle imaging velocimetry (PIV), fast-fourier transform-based alignment measurement and autocorrelation analysis.
On loaded DDCS, PHCF bidirectional migration and orientation along a single angle developed rapidly, colony patterning was stable and well-correlated and the ECM pattern reflected the cell pattern. On glass substrates, migration and orientation preferences developed more slowly, cellular patterns were metastable and were poorly correlated. On unloaded DDCS controls, colony patterns were also poorly correlated. The figure shows the evolution of cell migration speed and direction on the glass coverslip control in one location over a 12 day period.
The application of small mechanical-bias to the collagen substrate strongly altered the early migration behavior of individual cells leading to a stable emergent pattern. The synthesized ECM follows the emergent migration direction and orientation pattern suggesting that mechanical bias can control the format of matrix deposited by cells.
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