Retinal pigment epithelial cell culture

Abstract

Objective: Retinal pigment epithelial cells (RPEs), as the main cellular component of the preretinal membrane, whose excessive proliferation can lead to the development of proliferative vitreoretinopathy as interaction with other cellular components and intercellular substance. Here, we developed an effective method to culture RPEs so as to provide effective cell source for later experimental usage. Methods: The harvested of eye cup that retains the retinal pigment epithelium was gently washed three times with phosphate buffered saline (PBS), and was added with 0.02 mg/ml dispase enzyme, which was cut into small pieces and then placed in the incubator for digestion for about half an hour. Subsequently, gently blowing was performed multiple times to detach the RPEs from the retina to collect the RPEs suspension. After implantation into the culture dish, when the cells were grown and covered with 80-90% of the bottom of the bottle, digestion of the cells is carried out with 0.25% trypsin. After removing them from the wall, the digested cell suspension was collected, followed by addition of Dulbecco's modified Eagle medium/Nutrient Mixture F-12 (DMEM/F12) to stop digestion. The mixture was subject to centrifuge at 1000 r/min for 8 min, with the supernatant discarded, and then the cells located in bottom were passaged at 1:3 ratios. During the process, the morphology and viability were observed and cell identification were also performed. Results: Primary cultured cells began to adhere to the wall at 4 h, characterized with a flat and polygonal morphology, rich in pigment particles. On 3 days, there was a significant increase in cell numbers accompanied by cluster formation, resulting in a stabilized cobblestone-like appearance. By 5 days, cells elongated and formed dense clusters. Growth curve analysis revealed robust proliferation at 3 and 5 days. Immunofluorescence staining confirmed that over 90% of the cells were positive for RPE-65, indicating successful isolation and culture of pure rat RPEs. Conclusions: The RPEs cultured by the above method are in good growth and it can be used for further experimental studies.