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B. V. Stanzel, D. J. Strick, F. Thieltges, M. F. Marmor; Fibrillar vs. Smooth: Influence of Surface Characteristics on Cultured Human RPE Morphology and Protein Expression. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1834.
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© ARVO (1962-2015); The Authors (2016-present)
To develop tissue engineering strategies for the RPE using Bruch’s membrane prosthetics to treat retinal degenerations, including AMD. Common cell culture substrates are smooth surfaces. Nanofibers mimic the surface structure of native basement membranes. We explored the influence of both surface topologies on long-term differentiation of cultured human, and in particular, aged RPE.
Cell floaters of fetal and adult human RPE cells harvested by the Hu& Bok method were used for all experiments. 2.5x105/ cm2 cells were seeded on uncoated tissue culture treated plastic (PL), a smooth surface, or uncoated electrospun polyamide nanofibers (NF), which is fibrillar surface. Culture morphologies were monitored by phase contrast microscopy. RPE marker proteins were studied using immunofluorescence and immunoblotting at 4-6 weeks post confluence.
Fetal RPE, and RPE from a 23 year old donor developed uniform hexagonal morphology with interspersed fluid domes on both PL and NF. Cultures from an elderly (90 year-old) RPE failed to differentiate effectively on PL, but did show uniform hexagonal morphology with small fluid domes on NF. Gradual re-pigmentation was observed only in fetal RPE within the observation interval of 6 weeks. All cultures had positive immunofluorescence for Pancytokeratin, ZO-1 and RPE65 on NF. Claudin-3 expression was shown in fetal cells on both substrates. In addition, western blot analysis confirmed the expression of RPE65 in fetal and elderly RPE on NF.
NF appeared to be a superior substrate for aged RPE differentiation in our experiments with a floater cell line of one elderly eye pair. The fibrillar surface topology of EPN may help to rejuvenate aged RPE, thus making it a promising component for BM prosthetics. Further evaluation of EPN is currently underway.
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