Human retinal pigment epithelium expressed tight junction markers in spheroid cultures

Abstract

Abstract Purpose To determine if spheroid cultures generated from human prenatal retinal pigment epithelium (RPE) express markers for mature RPE and tight‐junction proteins and secrete components of the Bruchs membrane. Methods RPE was dissected from human prenatal donor eyes, chopped into 200 µm sections and place in suspension culture in serum‐free defined medium containing the commercially formulated supplement B27. Cultures were grown as spherical aggregate suspensions. At various times darkly spheroids were removed from the suspension and fixed with 4% PFA for one hour and cryoprotected with sequential sucrose solution. The spheroids were embedded in OCT, cut in 16 µm cryosections, and stained for different RPE and membrane markers including RPE65, CRALBP, PEDF, Occludin, Ezrin, Zo‐1 and laminin. EM was also performed. Results Inside the spheroids, RPE cells were located around a central space containing secreted laminin without a clear structure. Cells expressed tight‐junctions markers including ZO‐1, ezrin and occludin. Some of the cells had intracytoplasmic expression on CRALBP, RPE65 and PEDF. Long term culture spheroids showed a decrease in the number of RPE cells and increased pigment and debris. When examined with EM, RPE cells were located over a layer of fibroblasts, showing microvilli towards the outside of the spheroid and intracellular pigment. Conclusion Prenatal RPE maintained as spheriods show some RPE mature markers expression as well as tight junction markers. The cells are able to secrete laminin, an important component of the Bruch membrane.