Amniotic membrane maintains the phenotype of rabbit retinal pigment epithelial cells in culture

Primary RPE plastic cultures were established from freshly enucleated Dutch belted rabbit eyes in DMEM/F12 containing 0·1 mm Ca++ and 10% dialysed FBS. Upon subconfluence, cells were subcultured at 5000-9000 cells cm−2 in the above-mentioned culture medium on intact AM (iAM), epithelially denuded AM (dAM) or plastic. After confluence, the Ca++ concentration in the medium was increased to 1·8 mm for 4 weeks. Growth and morphology were monitored by phase contrast microscopy, and the phenotype by immunostaining with antibodies against cytokeratin 18, tight junction protein ZO-1, and RPE65 protein, and by transepithelial resistance (TER) measurement. Immunostaining to cytokeratin 18 confirmed the epithelial origin of isolated cells in both primary culture and subcultures. Compared to plastic cultures, RPE increased pigmentation within 24 hr after seeding on AM, with iAM being more pronounced than dAM. RPE adopted a hexagonal epithelial phenotype with more organised pigmentation, strong expression of ZO-1 and RPE65, and a significantly higher TER 4 weeks after Ca++ switch on dAM. Our results indicate that AM may be used as a basement membrane-containing matrix to maintain RPE phenotype in vitro, and may facilitate subsequent transplantation to treat ARMD.