Primary Culture and Identification of Rat Glomerular Podocytes

Objective To establish an easy and feasible method for primary culture and identification of rat glomerular podocytes. Methods Glomeruli from Sprague-Dawley(SD) rats weighing 60-100 gram we re isolated by the method of different size combination of screen. Isolated glomeruli were appropriately digested with 2 g/L type Ⅳ collagenase and cultured in 25 cm² plastic flask coated with rat tail collagen in K1-3T3 medium with ITS -X (containing insulin-transferrin-selenium). Subculture of primary cultured epithelial cells was performed at 9-10 days after implantation of collagenase d igested glomeruli. Podocytes were identified by the morphology study with scanni ng el ectron microscope and inverted microscope, as well as the immunohistochemistry s taining (SP methods) study for the expression of keratin, desmin and Wilms’ tum or suppressor-1 (WT-1). Results Epithelial cells outgrowth fr om isolated glomeruli appeared after 3 days primary culture and grew to confluen ce with cobblestone-appearance at 9-10 days. These cobblestone cells were subc ul tured at this point and gradually conversed into large, flat arborized cells wit h well-developed processes and microvilli. These arborized cells were negative expression with desmin staining and showed positive expression of cytokeratin an d WT-1, which indicated that they were podocytes. Conclusion Implantating collagenase digested-glomeruli is an easy and feasible metho d for primary culture of rat glomerular podocytes. WT-1 may serve as a good mar ker to identify rat glomerular podocytes.