Isolation, Purification, and Cultivation of Primary Retinal Microvascular Pericytes: A Novel Model Using Rats

Abstract

To isolate, purify, and cultivate primary retinal microvascular pericytes (RMPs) from rats to facilitate the study of their properties in vitro. Primary RMPs were isolated from weanling rats by mechanical morcel and collagenase digestion, and purified by a step-wise combination of selective medium with different glucose concentrations, medium exchange, and partial enzymatic digestion. Morphology of RMPs was assessed by phase contrast microscopy. Further characterization was analyzed by immunofluorescence. Functional assay was evaluated by the pericytes- endothelial cells (ECs) coculture system. Retinal microvascular pericytes migrated out of microvascular fragments after 24-48 hours of plating and reached subconfluence on days 14-16. The cells showed typical pericyte morphology with large irregular triangular cell bodies and multiple long processes, and uniformly expressed the cellular markers α-SMA, PDGFR-β, NG2 and desmin, but were negative for vWF, GS, GFAP and SMMHC. Ninety-nine percent of the cell population had double positive staining for α-SMA and PDGFR-β. In the coculture system, RMPs can directly contact ECs and move together to form the capillary-like cords. Retinal microvascular pericytes can be readily obtained by our method. We report the first cultivation of primary RMPs from rats and establish a simple method for their isolation and purification.