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I. Polzer, L. Paucz, A. Wedrich; Behavior of hTERT-RPE Cells Grown on Cryopreserved Amniotic Membrane in vitro . Invest. Ophthalmol. Vis. Sci. 2003;44(13):379.
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Purpose:to investigate conditions promoting physiological differentiation and behavior of human telomerase (hTERT) immortalised RPE cells grown on cryopreserved (50% glycerol in Dulbecco's MEM, stored at -170°C)amniotic membrane (cAM) in vitro. Methods:pure primary culture of human RPE cells (hRPE) from healthy donor eyes were stably transfected with the catalytic subunit of hTERT. The selection of pure hTERT immortal RPE cells was achieved by culturing transfected cells in phosphomycin-containing medium. The hTERT-RPE cells were seeded on cryopreserved amniotic membranes and maintained at confluence in retinal conditioned serum-free medium under stimulation with growth factors. Morphologic, immunocytochemical, immunofluorescent, confocal microscopic and photometric analysis were performed. Results:hTERT-RPE cells grown on cAM differentiated after reaching the confluence to growth arrested, confluent sheets of melanized polygonal cells in the absence of stimulation within 4 or 6 weeks. The pattern of pancytokeratin, S-100 and vimentin staining in hTERT-RPE cells was identical to that of primary hRPE. GFAP staining was negative in both. In comparison to primary hRPE cells, hTERT cells had increased replicative potential and decreased expression of senescence-associated beta-galactosidase activity, increased density and expression of tight junction zones as proved by ZO-1 antibody staining, as well as increased cellular melanin contain if grown on cAM. Conclusions: For their high replicative capacity and physiological behavior, hTERT-RPE cells grown on cAM provide a good experimental system to study the behavior and functional properties of RPE cells under various experimental conditions in vitro.
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