Primary culture and identification of human glomerular podocytes

Objective To establish a repeatable and feasible method for culturing human glomerular podocytes in vitro. Methods The renal epithelial tissues were obtained from the tumor-free pole of adults' kidneys after kidney tumor resection. Decapsulated human cortical slices were pressed through a series of stainless steel sieves (sieving method) with increasing pore sizes of 220 to 450 μm in a sterile, clean environment; as a final step, the glomeruli were collected on a 125 μm sieve and then cultured in 25 cm2 flasks of which bottoms were soaked by RPMI 1640 medium with 10% fetal bovine serum (FBS) in advance. Made the flasks upside down (explant method); 4 hours later, added medium and normally placed them. Cells were kept at 37 ℃ in a humid atmosphere under 5% CO2. Glomerular podocytes cellular markers as nephrin, Wilms tumor protein(WT-1), factor Ⅷ, vimentin and cytokeratin were analyzed by morphology and indirect immunofluorescence staining method, respectively. The purity of podocytes were determine by flow cytometry technology. Results Most of the glomeruli adhered to the wall of culture dish on the 3 rd day. Almost all glomeruli adhered on the 5 th day with a few climbed out polygonal cells which lost both primary and foot processes and appeared as cobblestones. A larger number of cobblestone-like cells which exhibited strong proliferative activity outgrowth from nearly every glomerular around from 7 th to 10 th day. Then the cells were digested by trypsin differences digestion method to remove fibroblast and subcultured. In subculturing, podocytes differentiate into other phenotypes which were large, branched, binucleated and exhibited no proliferative activity. It was observed by immunofluorescent staining that the cells were in line with characteristics of podocytes and expressed WT-1 and nephrin, but not the factor Ⅷ, vimentin and cytokeratin with no pollution of endothelial cells, mesangial cells and parietal epithelial cells. Furthermore, purity of podocytes was 98.3% checked by flow cytometry analysis. Conclusion It is simple and highly effective to culture primary cultured glomerular podocytes by combined sieving and explant method. Key words: Podocytes; Cell culture techniques; Adult