Background Retinal Müller cells are important gliocytcs and the source of retinal stem cells.Researching the biological behavior of Müller cells is of important significance to the study on retinal physiopathological process and stem cell therapy of retinal diseases.To establish a stable culture method of Müller cells is a solid basis of relative basic research.Objective This study was to establish a simple and stable method of isolation and culture of human retinal Müller cells and provide sufficient and high-quality Müller cell source.Methods Human retinal Müller cells were isolated from healthy human donor eyes.The mixture solution of hyaluronidase (100 U) and 0.25％ trypsin were used to digest chopped retinal tissue.The DMEM/F12 medium with 20％ fetal bovine serum (FBS) was added to stop the digestion process.RPMI1640 medium with 20％ FBS was used to culture the cell for 72 hours and then replaced the half medium.The cells were passaged by the RPMI1640 medium with 20％ FBS.The morphology of the cells were examied under the optical microscope,and the expressions of glial fibrillary acidic protein (GFAP),a marker of gliocytes,and glutamine synthetase (GS),a special marker of retinal Müller cells,were detected by immunochemistry and immunofluorescence technology.Results Human retinal Müller cells were successfully isolated by enzyme mixture solution of hyaluronidase (100 U) and O.25％ trypsin.The cells were adherent to walls 24 hours after primary culture and completely merged 9-10 days after culture.The cells showed oval in shape with abundant cytoplasm,and a part of cells presented with cone-shaped bulge bilaterally and ectasia in the posterior containing large nuclei.After cells passage,the cells were enlarged and grew toward polygonal shape.The positive expression of GFAP was observed in more than 95％ cells and strongly positive expression of GS was observed in more than 90％ cells by immunohistochemstry and immunofluorescent staining.Conclusions Human retinal Müller cells can be successfully isolated by hyaluronidase combined with trypsin digestion.Abundent and pure human retinal Müller cells can be obtained by successively using RPMI1640 medium with 20％ FBS and 10％ FBS.