Full Thickness Retinal Pigment Epithelium Explants Proliferate Into Epithelial Monolayers on Synthetic Bruch’s Membrane Substitutes

Abstract

Abstract: : Purpose: Investigators have been trying to culture RPE and IPE for potential implantation as a means of treating age–related macular degeneration (AMD). It has been difficult to grow cultures to take on proper epithelial characteristics. In this study, we use a new approach by harvesting full thickness RPE/Choroid explants as a nidus of RPE growth on synthetic Bruch’s membrane substitutes for potential autologous transplantation. Methods: Whole thickness RPE–Choroid sheets were harvested from adult New Zealand Red rabbits and cadaveric human eyes. Eyes were sterilized in 50% betadine then rinsed in sterile balanced saline solution. The anterior segment and vitreous was removed. A retinal detachment was created using hydrodissection to expose the underlying RPE. A 1 mm by 1 mm full–thickness block of choroid and RPE was removed and placed onto cellulose acetate (CA) or polycarbonate (PC) membranes. These were incubated in RPE media (DMEM/F–12 with 1% FBS and 1% penicillin/streptomycin) at 37º C. Growth of the RPE cells on CA and PC membranes were compared to seeded human cell line RPE cells (aRPE–19) and growth on mylar plastic. Growth was analyzed by light microscopy, live/dead staining, and immunohistochemistry. Results: On all surfaces, a robust growth of hexagonal epithelioid cells spread in a monolayer arising from the RPE/Choroid explants (either human or rabbit eyes). There did not appear to be a mixture of any other cellular types. These proliferating RPE cells showed great viability by live/dead staining, and they stained positively for immunohistochemical markers ZO–1 for tight junctions, DAPI for nuclei, and phalloidin for actin filaments, in a fashion similar to the known aRE–19 cell line. Conclusions: Extensive RPE cell monolayers can be cultured on Bruch’s membrane substitutes by using full thickness RPE/Choroid explants. This technique for harvesting RPE may have advantages over other more complicated methods. RPE monolayers grown on synthetic Bruch’s membrane might be of benefit for autologous cell transplantation for patients with age–related macular degeneration

Keywords: retinal pigment epithelium • choroid • Bruch's membrane