Cell culture of human RPE biopsies obtained during vitrectomy

Abstract

Abstract: : Purpose: Autologous RPE transplantation may become a treatment alternative for some retinal degenerations including AMD. In our previously described technique in patients with fCNV secondary to AMD, a RPE cell aspirate is transplanted subfoveally after membrane excision. The authors attempted to culture such aspirates to clarify the potential of these aged human RPE cells as an autologous graft. Methods: RPE biopsies were obtained from patients intended for autologous RPE transplantation for fCNV secondary to AMD, which due to low RPE transplant volume or hemorrhage served as controls and therefore received only membrane excision. After vitrectomy (PPV), the RPE was aspirated subretinally from 2–4 disc diameters (DD) with a special canula. A total 14 biopsies were obtained. The following substrates were seeded: TC plastic, Thermanox culture inserts, hexagonally imprinted Poly–Vinyl Alcohol (PVA) on Thermanox culture inserts and bovine corneal endothelial cell matrix (BCECM). Low Ca⁺⁺ DMEM/F12 medium was used for some plastic cultures. In another set of plastic cultures, in thermanox cultures with and without hexagonal PVA imprint a low Ca⁺⁺ DMEM/F12 medium supplemented with 1ng/ml bFGF was used. RPE biopsies on BCECM were cultured in a hormonally defined medium. Confluent cultures were switched to high Ca⁺⁺ or passaged and screened for RPE differentiation markers by immunofluorescence. Results: RPE biopsies showed viability of 70–80%. The aspirate contained single cells and sheets of RPE, red blood cells, photoreceptor outer segments, pieces of neural retina, and choriocapillaris/RPE patches. Between 250 to 1800 biopsied RPE cells were seeded in culture. Cultures on TC plastic using low Ca⁺⁺ showed survival of 2–4 weeks. Adding bFGF to the cultures improved survival of a few cells up to 3months in 35.7% of cases. Some cultures on hexagonally imprinted PVA on thermanox and BCECM yielded confluent cultures with epithelial morphology. Culture success also appeared related to higher seeding densities. Immunofluorescence for cytokeratins showed altered or lost expression in biopsies compared to enzymatically harvested human RPE cultures. Conclusions: Culture of transvitreally biopsied human RPE was possible in a few instances and required more sophisticated culture conditions. The present study suggests some preserved regenerative capacity for aged human RPE in AMD patients.

Keywords: retinal pigment epithelium • transplantation • retinal culture