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S. V. Patel, L. A. Bachman, C. R. Hann, M. P. Fautsch; Attachment of Cultured Human Corneal Endothelial Cells to Corneal Stroma. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3946.
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To investigate whether cultured human corneal endothelial cells (HCECs) can attach to the posterior stroma of recipient corneas in vitro.
Human corneas, unsuitable for transplantation and stored in Optisol-GS preservation medium, provided "donor" endothelial cells for culture or became "recipient" corneas. Donor HCECs were harvested and cultured in medium containing 8% fetal bovine serum supplemented with EGF, FGF and NGF. Confluent HCECs were incubated overnight with 0.9-µm superparamagnetic microspheres (SPMs) at 500 SPMs per cell plated. In recipient corneas, Descemet’s membrane and endothelium were stripped to expose posterior stroma (n=4). Donor HCECs (~350,000 cells) were washed, labeled with DiI (live cell fluorescent stain), and transferred to the concave posterior surface of recipient corneas (with epithelial surface down). Recipient corneas with donor HCECs were placed over a 3390-Gauss magnet or over a non-magnetic disc (control), and incubated in culture medium for 3-4 days. Recipient corneas were rinsed thoroughly and cultured for an additional 4-21 days. Corneas were examined by fluorescence, light, and electron microscopy to determine whether donor HCECs that contained SPMs were attached to the stroma.
Donor HCECs were successfully cultured in vitro and formed a near-hexagonal monolayer similar to corneal endothelium in vivo. DiI-labeled (donor) HCECs were detected on the posterior surface of all recipient corneas by fluorescence microscopy. Light microscopy showed attachment of endothelial cells, labeled with SPMs, to posterior stroma when the recipient was placed over the magnet, but not when it was placed over the non-magnetic control disc. Transmission electron microscopy confirmed donor endothelial cell attachment to recipient corneal stroma.
Cultured donor HCECs can attach to recipient posterior stroma in vitro; further studies are needed to determine the mechanism of attachment and the role of magnetic attraction in enhancing attachment. Improving the efficacy of HCEC attachment to corneal stroma will be important for developing methods of endothelial cell transplantation for diseases which require removal of host Descemet’s membrane, such as Fuchs’ endothelial dystrophy.
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