June 2015
Volume 56, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2015
Long-term, Live-Imaging of the Mechanodynamics of Primary Human Corneal Fibroblasts: From Initial Seeding to Matrix Production
Author Affiliations & Notes
  • Jeffrey Ruberti
    Bioengineering, Northeastern University, Boston, MA
    Schepen's Eye Research Institute, Boston, MA
  • Ramin Zareian
    Bioengineering, University of California Berkeley, Berkeley, CA
  • Dimitrios Karamichos
    Ophthalmology, University of Oklahoma Health Sciences Center, Oklahoma City, OK
  • Dhananjay Tambe
    Mechanical Engineering, University of South Alabama, Mobile, AL
  • Monica Susilo
    Bioengineering, Northeastern University, Boston, MA
  • Jeffrey Paten
    Draper Laboratory, Cambridge, MA
  • Nima Saeidi
    Massachusetts General Hospital, Boston, MA
  • James D Zieske
    Schepen's Eye Research Institute, Boston, MA
  • Footnotes
    Commercial Relationships Jeffrey Ruberti, None; Ramin Zareian, None; Dimitrios Karamichos, None; Dhananjay Tambe, None; Monica Susilo, None; Jeffrey Paten, None; Nima Saeidi, None; James Zieske, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2015, Vol.56, 1934. doi:
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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)

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Abstract
 
Purpose
 

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.

 
Methods
 

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.

 
Results
 

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.

 
Conclusions
 

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.  

 
Cell colony migration velocity rose plots showing the change in the velocity patterns over 12 days. Plots were selected from days 1, 6 and 12 and are based on PIV data from one location on the coverslip.
 
Cell colony migration velocity rose plots showing the change in the velocity patterns over 12 days. Plots were selected from days 1, 6 and 12 and are based on PIV data from one location on the coverslip.

 
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