Purchase this article with an account.
Ramin Zareian, Dimitrios Karamichos, Jeffrey A. Paten, James D. Zieske, Jeffrey W. Ruberti; Live, Long-Term Observation of the Mechanobiology of Confluent Primary Human Corneal Fibroblast Cultures Subjected to Applied Force. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1529.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
: It has now been clearly established that mechanics plays a critical role in the initial templating, subsequent growth and maintenance of mesenchymal tissue. Mechanics strongly influences both the differentiation state and general behavior of mesenchymal cells (stem and fibroblasts). In addition, the interaction systems of associated molecules have shown exquisite mechanosensitvity. We have employed a custom mechanobioreactor to observe changes in the dynamic organizational behavior of Primary Human Corneal Fibroblasts (PHCFs) exposed to uniaxial load.
Our device was sterilized under aseptic conditions and loaded with a dense, disorganized collagen scaffold (DDCS) comprising type I bovine collagen. PHCFs from human donors were extracted, cultured, and seeded into the device where they grew both on the DDCS and on the coverslip. The culture medium was continuously fed to the cells at a fixed flow rate (8ul/min). After reaching confluence the DDCS culture was exposed to a mechanical stretch of 6% (N=4). In one of the four experiments PHCFs were exposed to the live nuclear stain to permit cell tracking. Fluorescent and DIC images were taken every hour for two weeks. PIV (Particle Imaging Velocimetry) was used to track cell while FFT (Fast Fourier Transform) methods were used directly on the DIC images to extract local and global orientation of cells.
All experiments exhibited similar organizational behavior indicating that the live stain did not alter the culture significantly. Continuous movies reveal a highly dynamic culture system with rapidly moving PHCFs which initially display random orientation. Confluence led to local organization both on the DDCS and on the glass coverslip. However, loading the DDCS led to consistent global alignment of the PHCF at a relatively fixed angle to the applied force.
Mechanical load has a profound effect on the migration and organizational behavior of PHCFs in culture and could likely be used to influence the organization of synthesized matrix.A) Live nuclear stain of PHCFs, green and white arrows show the direction of motion and load respectively, B) Median velocity angle of cells on substrate (green) and coverslip (red) C) FFT analysis of the cells.
This PDF is available to Subscribers Only