Figure 1

The pipeline for establishing conditionally immortalized MPCs. (A) To obtain podocytes for establishing a conditionally immortalized MPC model, glomeruli were harvested from suckling mice. Kidneys were minced to 1-mm³ pieces in a 6-cm cell culture dish. The tissues were digested in collagenase solution. The collagenase-digested tissues were gently pressed through a 70-µm cell strainer, and the glomeruli were cultured at 37 °C. (B) Two days later, outgrowing cells appeared as a monolayer around the individual glomeruli. Trypsin-digested cells were passed through a 40 µm cell strainer to remove the glomerular cores. The primary glomerular cells were cultured at 37 °C until the cell confluence reached 80%. (C) Podocytes were infected with lentivirus carrying SV40 tsA58 and the puromycin resistance gene. We performed limited dilutions in 96-well plates and identified and numbered individual cell wells using microscopy. (D) Thirteen monoclonal cell lines were successfully isolated. The 13 monoclonal cell lines were subjected to preliminary identification using RT-qPCR. A monoclonal podocyte line was used for further identification. Imaging materials were obtained from smart.servier.com. The scale is 100 µm.