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Timothy A Blenkinsop, Janmeet S Saini, Arvydas Maminishkis, Kapil Bharti, Qin Wan, Janine Davis, Sheldon S Miller, Sally Temple, Jeffrey Stern; Human Adult Retinal Pigment Epithelial Cultures Exhibit Key Physiological Characteristics of Native RPE Tissue. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2330.
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© ARVO (1962-2015); The Authors (2016-present)
Evaluate the physiology of adult human RPE cultured using a recently established protocol.
Adult human eyes were dissected, and RPE mechanically removed in sheets and plated in tissue culture plates in a medium developed for the maintenance of cobblestone morphology. Physiology experiments were performed with previously described techniques. Standard techniques for immunohistochemistry, electron microscopy, electrophysiology, fluid transport, and cytokine secretion measurement by ELISA were used.
We demonstrate our method of adult human culture produces at least 6 x 107 RPE from one donor. The cultures possess polarized expression of proteins essential for RPE function. Electron micrograph images demonstrate normal morphology including microvilli, tight junctions, apically localized pigmentation, basolaterally localized nucleus and more. These cultures maintain in vivo transepithelial resistance, membrane potentials and fluid transport. Ionic perturbations and drug applications demonstrate normal localization of Na+, K+, and Cl- channels and normal responses to adrenergic and catecholaminergic stimulation. Gene expression data comparing adult human RPE to native RPE and fetal cultured RPE confirm the maintenance of RPE identity. Lastly, polarized cytokine secretion is similar to what is observed in other RPE model systems.
Adult human RPE cultures exhibiting morphological and electrophysiological characteristics as well as gene and protein expression patterns that mimic the native tissue may be a useful model to study RPE physiology, disease and transplantation.
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