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E. M. Bueno-Hoyos, N. Saeidi, J. W. Ruberti; Live Long-Term Monitoring of in vitro Matrix Synthesis and Organization by Bovine Corneal Fibroblasts. Invest. Ophthalmol. Vis. Sci. 2007;48(13):1499.
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To observe, using a non-invasive imaging method, the live in vitro secretion and organization of matrix by bovine corneal fibroblasts stimulated with ascorbic acid over long time periods.
4th passage bovine corneal fibroblasts cultivated for 2 weeks on glass coverslips under ascorbic acid stimulation were mounted onto a microincubation chamber slowly perfused with gassed culture medium. Time-lapse imaging was carried out via long-term live dynamic differential interference contrast (LLDDIC), which allowed simultaneous visualization of individual cells and their surrounding matrix. The LLDDIC system comprised a focal plane-stabilized (Perfect Focus®) TE2000U inverted microscope (60x 1.45 NA objective - Nikon, Melville, NY), a microincubation chamber (Bioptechs, Butler, PA) fitted with a syringe pump (PHD2000, Harvard Apparatus, Holliston, MA) and a Coolsnap EZ camera (Photometrics, Tucson, AZ). Cells within a target area were monitored by acquiring images every 2 minutes for 1 week. In addition, 3-D images were constructed by acquiring daily images at 0.1-µm increments in the z-direction. Movies of the motion and synthetic activity of individual cells were obtained. Quick Freeze Deep Etch (QFDE) imaging of the cells and matrix complemented the LLDDIC imaging. Briefly, the constructs were slam frozen onto a copper block at -196ºC (Cryogun - Delaware Diamond Knives, Dover, DE) and etched for 1 h in a customized CFE-40 freeze fracture/freeze etch apparatus (Cressington Scientific, Watford, UK). Samples were then low-angle rotary-shadowed with Pt/C and backed with carbon at a 90º angle. Replicas were cleaned in bleach, washed and picked up on 600-mesh grids for electron microscopy (JEOL JEM-1000).
A 4-µm layer of cells that remained viable and active as evidenced by numerous cell divisions and vigorous motion was monitored live for a 1-week period. Random motion and numerous cell-cell interactions were observed. The synthesis of matrix fibers was accompanied by a reduction in cell motion. Preliminary QFDE imaging detected two sets of fibers, approximately 100- and 30-nm in diameter. The 30-nm fibrils are thought to be collagen, the 100 nm fibrils were observed as extensions of the cell membrane. Type I collagen presence was verified via monoclonal antibody staining.
The LLDDIC imaging system was successfully utilized to monitor long-term in vitro fibroblast activity. LLDDIC coupled with our stimulated ECM synthesis model and QFDE imaging may elucidate how cells "stitch" together organized ECM.
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