Primary isolation and cultivation of retinal Müller cells in Sprague-Dawley rats

Abstract

Objective: Müller cells in the retina are one of the most important supporting cells in the retina, belonging to the category of glial cells. This study used gradient low concentration enzyme repeated digestion method to isolate Müller cells in the retina of Sprague-Dawley (SD) rats. Methods: Retinal Müller cells were harvested in retina from newborn SD rats. Initially, the retinal tissue was carefully dissected, digested with trypsin at 37°C for 15 minutes, and filtered. Following the centrifugation and resuspension in DMEM/F12 medium with 10% fetal bovine serum, cells were cultured in a gelatin-coated plate. After reaching confluence, cells were digested with low-concentration trypsin to improve purity. Immunofluorescence staining was performed using GFAP antibody staining and DAPI counterstain to confirm cell identity. Results: Retinal Müller cells began adhering and forming clusters within 24 hours, with confluence achieved by days 3-6. After subculturing, the cells exhibited uniform morphology, elongated shapes, and round/oval nuclei. Immunofluorescence on day 7 showed over 80% GFAP-positive reaction, confirming their glial nature and the successful isolation of Müller cells. Conclusion: This method is effective for obtaining a stable population of Müller cells, which can be used for further research on retina.