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Lori A. Bachman, Laura A. Hecker, Cindy K. Bahler, Bradley H. Holman, Cheryl R. Hann, Michael P. Fautsch, Sanjay V. Patel; Morphologic Characterization of Cultured Human Corneal Endothelial Cells after Transplantation to Human Corneal Stroma Ex Vivo. Invest. Ophthalmol. Vis. Sci. 2011;52(14):6458.
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© ARVO (1962-2015); The Authors (2016-present)
Previous studies by our laboratory have demonstrated that human corneal endothelial cells (HCECs) containing superparamagnetic particles (SPMs) can be successfully seeded to recipient human corneal stroma by using an external magnetic field. In this study we investigated modifications in the strength and duration of exposure to the magnetic field.
HCECs were harvested from donor corneas and cultured in monolayer in vitro. Confluent passage 2 HCECs were incubated overnight with 100 nm SPMs at 1000 SPMs per cell plated and labeled with CM-DiI. HCECs with SPMs (500,000-1,000,000 cells) were transferred to 6 fellow pairs of recipient human anterior segments in organ culture after stripping Descemet membrane and endothelium from the cornea. A 3855 or 4385 Gauss magnet was placed adjacent to the epithelium of one recipient cornea for 48-72 hours, whereas the fellow recipient cornea was not exposed to a magnetic field. After removal of the magnet, corneas were cultured for an additional 1-12 days before examination by fluorescence, light and scanning electron microscopy. Donor endothelial cell density was calculated from scanning electron or fluorescence confocal microscopy. Expression of cell junction-associated proteins ZO-1 and integrin β1 was analyzed by immunohistochemistry.
In all experiments, the presence of the magnetic field aided attraction of the cultured donor HCECs to the recipient stroma and facilitated the formation of a near-hexagonal monolayer of cells. HCECs forming the monolayer were confirmed as donor cells by CM-DiI labeling and Prussian blue staining for intracellular iron. Donor HCEC density in corneas exposed to the magnetic field was 2,377 ± 1,290 cells/mm2 (mean ± SD; range, 700-4450 cells/mm2). ZO-1 staining of intercellular junctions appeared punctate, and became more pronounced with longer duration of organ culture. Donor HCECs expressed integrin β1, which was localized to intercellular junctions and the interface between HCECs and recipient stroma.
Cultured HCECs can attach to the fibrillar collagen of recipient corneal stroma and form an intact monolayer with intercellular junctions. The strength and duration of exposure to the magnetic field might affect attachment and resulting donor endothelial cell density. Adhesion of HCECs directly to corneal stroma might involve integrin β1, which is known to bind fibrillar type I collagen.
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