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Ilene K. Sugino, Qian Sun, Marco A. Zarbin; BCE-Conditioned Media Stimulates RPE Extracellular Matrix (ECM) Deposition. Invest. Ophthalmol. Vis. Sci. 2011;52(14):6063.
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Bovine corneal endothelial cell-conditioned media (BCE-CM) enhances cell survival on aged and AMD Bruch's membrane while a medium routinely used in RPE cell culture ("RPE media") supports cells on these same surfaces to a limited degree. We have shown previously that cell survival can be enhanced on aged Bruch's membrane by resurfacing Bruch's membrane with a cell-deposited ECM. Bovine corneal endothelial cell-conditioned media (BCE-CM) includes soluble ECM components which could stimulate increased RPE ECM deposition and thereby enhance cell survival in a similar fashion. We investigated whether RPE ECM deposition was increased following culture in BCE-CM.
Bruch's membrane explants from AMD and age-matched non-AMD donors (3 donor pairs each) were seeded with fetal RPE and cultured for 21 days in BCE-CM or RPE media. Calcein imaging was performed prior to cell removal with NH4OH to determine surface coverage by RPE. After cell removal, explants were bisected and the surface of Bruch's membrane was immunostained for laminin and collagen IV (one half) or fibronectin and laminin (other half). Explants were evaluated with confocal microscopy then processed for scanning electron microscopy (SEM) evaluation.
Laminin, collagen IV, and fibronectin labeling on explants cultured in BCE-CM was extensive and complex while explants cultured in RPE media showed no or sparse labeling. Laminin, collagen IV, and fibronectin labeled a network of fibers, with some colocalization of laminin and collagen IV. Scanning electron microscopic evaluation of the explants showed bare ICL or a few strands on the surface of the bare ICL in explants cultured in RPE media. On explants cultured in BCE-CM, a network of fibers was observed, varying from a complex mesh of fibers to a fairly continuous cover overlying the ICL.
The stimulation of increased ECM deposition by culturing RPE in BCE-CM suggests a mechanism by which RPE survival is enhanced on aged and AMD Bruch's membrane.
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