Abstract: 【Abstract】 Objective To establish a reliable method for primary culture of rat retinal microvascular endothelial cells (RRMEC) in vitro in order to study the basic pathogenesis of retinal neovascularization disease. Design Experimental study. Participants Rat retinal microvascular endothelial cells. Methods With the isolation of active retinal blood vessels and enzyme digestion of retinal tissue, RRMECs were cultured in RPMI-1640 supplemented with fetal bovine serum, and endothelial cell growth supplement (ECGs). The RRMECs were observed under microscope. Immunocytochemistry was also performed using a monoclonal antibody against Factor VIII and platelet endothelial cell adhesion molecule (PECAM-1, CD31) for identification. Main Outcome Measures RRMEC morphology and fluorescence staining. Results RRMECs in primary culture attached the walls and scattered in monolayer arrangement with clear boundary, mostly in fusiform, partly in cobblestone-like growth. RRMECs began to confluence within 3-5 days. The purity of RRMEC was high. The rate of  positive staining for Factor VIII (fluorescence staining perinuclear) and CD31 (fluorescence staining in cytoplasm) was 95%. Conclusions RRMECs with high purity can be obtained by isolation of retinal blood vessels, digestion with trypsin and collagenase, and use of ECGs and gelatin-coated bottles, which is a repeatable better method. (Ophthalmol CHN, 2012, 21: 261-263)

Key words: retinal microvascular endothelial cells, rats, cell culture