Confluent Monolayers of Cultured Human Fetal Retinal Pigment Epithelium (hfRPE) Exhibit Morphology and Physiology of Native Tissue

Abstract

Abstract: : Purpose:To develop a reproducible method for culturing hfRPE cells that produce confluent monolayers that exhibit morphology, physiology, polarity and protein expression paterns similar to native hfRPE. Methods: The research followed the tenets of the Declaration of Helsinki and the institutional review board. Fetal eyes were obtained by an independent procurer (ABR; Alameda,CA) and delivered using overnight priority service. Eyes were dissected upon delivery and RPE cell sheet mechanically separated from choroid and cultured in a specifically designed medium comprised entirely of commercially available components. All physiology experiments were carried out using previously described techniques (Maminishkis A., et al., IOVS, v.43: 3555, 2002). Standard techniques were used for immunohistochemisry, electron microscopy, and ELISA. Results: Confluent monolayers of RPE cell cultures exhibit epithelial morphology and heavy pigmentation and electron microscopy shows extensive apical membrane microvilli and tight junction (TJ) complexes. TJ complexes were identified using immunofluorescence labeling of ZO–1 and occludin. The mean transepithelial potential (TEP) was 2.6 ± 0.8 mV, apical positive and the mean transepithelial resistance (R_t) was 501 ± 138 Ω·cm² (mean ± SD; n = 35). Addition of 100 µM ATP to the apical bath increased net fluid absorption from 13.6 ± 2.6 to 18.8 ± 4.6 µl·cm^–2·hr^–1 (mean ± SD; n = 4). We also found that VEGF secretion into the apical bath is half of the secretion to the basal bath (n = 10). In contrast, PEDF secretion is two–fold greater for the apical compared to the basal bath (n = 10). Conclusions: A new cell culture procedure has been developed that produces confluent primary hfRPE cultures with morphological and physiological characteristics of the native tissue. Pharmacological perturbations to the apical and basolateral membranes of these monolayers were studied using microelectrode, fluorescence, and fluid transport techniques. Epithelial polarity and function of these easily reproduced primary cultures closely resembled previously studied native hfRPE.

Keywords: retinal pigment epithelium • retinal culture • microscopy: light/fluorescence/immunohistochemistry