Growth and Differentiation of Rabbit RPE Cells on Cryopreserved Human Amniotic Membrane

Abstract

Abstract: : Purpose: The success of surgical removal of choroidal neovascularization (CNV) followed by transplantation of autologous retinal pigment epithelial cells (RPE) for age-related macular degeneration (ARMD) may be potentially limited due to permanent damage in Bruch’s membrane. We surmise amniotic membrane (AM) can be used as an alternative basement membrane-containing ECM to support RPE growth and differentiation. Methods: Primary RPE cultures were established from freshly enucleated Dutch belted rabbit eyes in DMEM/ F12 containing 0.1 mM Ca⁺⁺, 10% dialyzed fetal bovine serum, 100 IU/ml penicillin, 100 µg/ml streptomycin and 0.5 µg/ml amphotericin B. Upon confluence, cells were subcultured at 5,000-9000 cells/cm² in the above culture medium on intact AM (iAM), epithelially-denuded AM (dAM) or plastic. Upon confluence, the Ca⁺⁺ concentration in the medium was increased to 1.8 mM for 3-4 weeks. Growth and morphology were monitored by phase contrast microscopy, and the phenotype by immunostaining with antibodies against keratin 18, tight junction ZO-1, and RPE-65 protein. Results: Immunostaining to keratin 18 confirmed the epithelial origin of isolated cells in both primary culture and subcultures. Growth rate was comparable on dAM and plastic, but was slower in iAM. Compared to plastic cultures, RPE increased pigmentation within 24 hr after seeding on AM, with iAM being more pronounced than dAM. On plastic and dAM, cells were mostly spindle-shaped, while those on iAM formed spheroid aggregates within 24 hr, and gradually adhered and flattened on iAM in one week. RPE adopted a hexagonal epithelial phenotype with more organized pigmentation and positive ZO-1 expression 2-3 weeks after Ca-elevation on dAM. Immunofluorescence staining confirmed strong expression of RPE-65 protein by RPE grown on dAM and iAM and sporadic positive expression on plastic 3 wks after Ca⁺⁺- elevation. Conclusions: Our results indicate that cryopreserved amniotic membrane may serve as an alternative basement membrane substrate to promote growth and proper differentiation of rabbit RPE in vitro. This culture system can potentially be modified to improve the success of human RPE transplantation for treating various retinal degenerative disorders such as ARMD involving RPE and Bruch’s membrane.

Keywords: retinal pigment epithelium • Bruch's membrane • age-related macular degeneration