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E. Chankiewitz, B. Bachmann, E. Blazejewska, U. Schloetzer-Schrehardt, G. Thumann, C. Rummelt, F. Kruse; A Novel Ultrathin Collagen Membrane for ex vivo-Expansion and Transplantation of Corneal Epithelial Cell Sheets. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1521.
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To investigate the suitability and biocompatibility of a defined ultrathin collagen matrix for ex vivo-expansion and transplantation of corneal epithelial cells sheets as a substitute for amniotic membrane.
Isolated human limbal epithelial stem cells were clonally enriched on 3T3 feeder cells and subcultivated on ultrathin (40 µm) collagen membranes composed of type I collagen (ResoFoil, Resorba, Germany) to establish multilayered corneal epithelial cell sheets. Cell adhesion and proliferation as well as cellular phenotype and differentiation were examined by light and electron microscopy, proliferation assay, and immunohistochemistry using antibodies against stem cell markers (p63, ABCG2, K15) and corneal differentiation markers (K3, K12, Pax6) in vitro. Mechanical qualities and biocompatibility of collagen membranes were assessed after corneal transplantation in animal models following removal of the central epithelium and anterior stroma. Histological analysis of excised corneas was performed after various time points.
Collagen membranes supported adhesion, proliferation, and differentiation of limbal stem cells and formation of multilayered epithelial cell sheets. Cultured cells showed ultrastructural signs of differentiation and expressed K3, K12 as well as the transcription factor Pax6 throughout all cell layers, while stem cell markers were immunolocalized to basal cells only. Collagen membranes transplanted into the cornea of rabbits and mice provided adequate mechanical strength and stability without any signs of inflammation for up to 4 weeks after surgery. Corneal epithelial closure was observed after 5-6 days and corneal transparency was obtained within 2-3 weeks. Histological analysis revealed overgrowth of collagen membranes by corneal epithelium as well as repopulation by stromal keratocytes.
These data suggest that ultrathin collagen membranes facilitate ex vivo-expansion and transplantation of limbal progenitor cells and may therefore represent a defined alternative to amniotic membrane which is subject to biological variability.
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