The Petri Dish-N2B27 Culture Condition Maintains RPE Phenotype by Inhibiting Cell Proliferation and mTOR Activation.

Hui Lou,Xiaoyan Ji,Chunpin Lian,Xiaoyun Zhao,Guoxu Xu,Jingfa Zhang,Fanjun Shi,Sicheng Qian,Hui Wang,Liqun Chen

doi:10.1155/2020/4892978

Abstract

Objective To develop a method for the rapid isolation of rat RPE cells with high yield and maintain its epithelial state in modified culture system. Methods The eyeballs were incubated with dispase. The retina was isolated with RPE attached and cut into several pieces. Following a brief incubation in growth medium, large RPE sheets can be harvested rapidly. RPE cells were divided into four groups and cultured for several weeks, that is, (1) in cell culture dishes with 10% FBS containing medium (CC dish-FBS), (2) in petri dishes with 10% FBS containing medium (Petri dish-FBS), (3) in cell culture dishes with N2 and B27 containing medium (CC dish-N2B27), and (4) in petri dishes with N2 and B27 containing medium (Petri dish-N2B27). Morphological and biological characteristics were investigated using light microscopy, Q-PCR, and western blot. Results The retina would curl inwardly during the growth medium incubation period, releasing RPE sheets in the medium. Compared with low density group (5,000 cells/cm2), RPE cells plated at high density (15,000 cells/cm2) can maintain RPE morphology for a more extended period. Meanwhile, plating RPE cells at low density significantly reduced the expression of RPE cell type-specific genes (RPE65, CRALBP, and bestrophin) and increased the expression of EMT-related genes (N-cadherin, fibronectin, and α-SMA), in comparison with the samples from the high density group. The petri dish culture condition reduced cell adhesion and thus inhibited RPE cell proliferation. As compared with other culture conditions, RPE cells in the petri dish-N2B27 condition could maintain RPE phenotype with increased expression of RPE-specific genes and decreased expression of EMT-related genes. The AKT/mTOR pathway was also decreased in petri dish-N2B27 condition. Conclusion The current study provided an alternative method for easy isolation of RPE cells with high yield and maintenance of its epithelial morphology in the petri dish-N2B27 condition.

Highlights

Hui Lou,1 Chunpin Lian,2 Fanjun Shi,1 Liqun Chen,1 Sicheng Qian,1 Hui Wang,3 Xiaoyun Zhao,1 Xiaoyan Ji,1 Jingfa Zhang,4,5 and Guoxu Xu 1
It has been reported that cell density has a profound influence on retinal pigment epithelium (RPE) epithelial phenotype [18]. erefore, we investigated the effect of initial cell seeding density on the morphology and tissuespecific genes’ expression of RPE cells
RPE cells exert many essential functions in the visual cycle, and their dysfunction would lead to retinal degeneration and vision impairment. e RPE cell has an apical to basolateral polarity by structure, characterized by the presence of apical microvilli and basal infoldings. is, as well as its barrier function that inhibits drug molecules from passing from the blood to the retina, makes experimentation with RPE difficult in vivo. us, the primary RPE cell culture serves as a valuable in vitro model to study RPE transport, protein localization and function, and the side-effects of drugs on RPE cells

Summary

Objective

To develop a method for the rapid isolation of rat RPE cells with high yield and maintain its epithelial state in modified culture system. As compared with other culture conditions, RPE cells in the petri dish-N2B27 condition could maintain RPE phenotype with increased expression of RPE-specific genes and decreased expression of EMT-related genes. E current study provided an alternative method for easy isolation of RPE cells with high yield and maintenance of its epithelial morphology in the petri dish-N2B27 condition. ARPE-19 cells are often characterized by losing of RPE cell type-specific features and acquiring mesenchymal cell-like properties They possessed low transepithelial electrical resistance and decreased expression of RPE-selective genes [6, 7], limiting its potential use to unveil the pathogenesis of RD. E present study showed that this could be an alternative method with easy manipulation for RPE isolation and in vitro culture, facilitating its further study for the pathogenesis of RD

Materials and Methods

Results

Discussion

Disclosure