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F. Thieltges, C. Strack, F. G. Holz, B. V. Stanzel; Nanofibrillar vs. Smooth: Influence of Surface Characteristics on Cultured Human RPE Proliferation. Invest. Ophthalmol. Vis. Sci. 2009;50(13):6228.
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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 compared the influence of these surface topology variants on proliferation in cultured RPE.
Human RPE cells from fetuses, a 23 and 90 year old donor were cultured in Hu & Bok medium. Cell growth was stimulated with medium containing 10% serum and then cell cycle synchronized by serum starvation for 24h. Cells were replated at 104 cells/cm2 on either tissue culture treated polystyrene (PS), a smooth surface, or uncoated electrospun polyamide nanofibers (EPN), a fibrillar surface. Growth curves were established with Trypan blue staining and automated counting of representative high power fields (HPF) with ImageJ software. A double label technique for anti-BrdU and DAPI was developed to assess growth fractions. To define S-phase lengths on PS and EPN, cells were BrdU pulsed for increasing time intervals at 48-72h post seeding.
Proliferating cells on EPN showed polygonal morphology with colony forming distribution compared to a rather spindle-shaped appearance and random distribution on PS. Cell division on EPN started earlier in all donor ages. Proliferation rates of fetal RPE during early log phase were equal on both substrates, but increased with adult cells on EPN in pilot experiments. Final cell densities per HPF at confluence in fetal cultures were lower on EPN compared to PS, inverse to pilot data in adult cells. Pretreatment with 2N HCl for 10 minutes produced double labeling with anti-BrdU on a DAPI background, differing in anti-BrdU signal intensities. During early log phase in fetal cultures, total BrdU positive cells (growth fraction) on PS were 80% (SD 15) versus 69% (SD 14) on EPN. With increasing time intervals of the BrdU pulse, more BrdU positive nuclei and a broader signal spectrum was observed. In fetal cultures, short pulses yielded more BrdU positive cells on EPN than on PS.
A nanofibrillar surface induces an epitheloid growth pattern, while on a smooth surface it is spindle-shaped. Moreover, nanofibers stimulate faster BrdU incorporation, suggesting a shorter S-phase length when compared to a smooth surface. The nanofibrillar surface topology of EPN appears to rejuvenate adult RPE, thus making it a promising component for BM prosthetics. Further studies with nanofibers are underway.
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