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BV Stanzel, K-H Huemer, PK Ahnelt, H Feichtinger, S Binder; Minimal Plating Density of Adult Human RPE Cells Using a Ca++ Adjusted DMEM/F12 Culture Medium . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3437.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Combined subretinal surgery and autologous retinal pigment epithelial (RPE) transplantation in choroidal neovascularization (CNV) due to AMD is a promising therapy. However, repopulation of large defects as created after excision of a CNV may require intermittent in vitro expansion of autologous RPE. Aim of the present study was to define minimal RPE plating density and optimal culture conditions in a model system. Methods: RPE cultures were established from three human cadaveric eyes (10-15h post mortem) from donors for corneal transplants. 3rd passage cells were used for the plating experiments in 96 well plates. Cells were grown on plastic or laminin coated wells in unmodified DMEM/F12 and, alternatively, in a low Ca++ (0.1mM) DMEM/F12, each supplemented with 10% dialyzed FBS. In case of initiation with low Ca++, ion concentration was increased to 1.8mM after confluency. A Buerker-Tuerk chamber was used for the cell counts. Results: A critical number of approximately 1500 cells per well (0.35cm2) was needed to reach confluency after a period of 28 days or longer. An increase of initial cell numbers to 2500 significantly shortened the time to grow confluent RPE monolayers to 10-14 days. Modifications of Ca++ concentrations proved to be essential for successful initiation and propagation of RPE cell growth. Plastic was superior to laminin coating. All confluent cultures exhibited epithelial morphology, regardless of cell density. Differentiation increased gradually over several weeks post confluency with partial remelanization and discernible phase bright cell borders. Immunocytochemistry for pancytokeratin as well as cytokeratin subtypes 8 and 18 was positive for all cells. Conclusion: A critical plating density of 1500 cells per 96-well, modulations of the Ca++ concentration of the culture medium, and adherence to plastic were essential requirements in a model system for the in vitro expansion of primarily low numbers of RPE. Further studies are needed to transfer these findings to the initiation and propagation of primary RPE cultures.
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