Type 1 collagen coated flasks

Human urine derived podocyte-like epithelial cells (HUPECs, termed podocytes for simplicity) isolated from a healthy volunteer were described previously [28] (link). The podocytes carried a temperature-sensitive variant of the simian virus (SV40) large tumor antigen and were cultured and maintained, as previously described [28] (link). Briefly, podocytes were grown on type I collagen-coated flasks (Fisher Scientific, Rochester, NY) at 33°C (growth permissive conditions) in culture medium (RPMI 1640 medium (Sigma), 10% fetal bovine serum (Hyclone, Logan, UT), 15 mM HEPES, 5 mM L-glutamine, 100 U/ml penicillin, 100 U/ml streptomycin; 5% CO₂ atmosphere). After reaching confluency, cells were seeded onto 35 mm type I collagen-coated dishes and maintained at 37°C (growth restrictive conditions) for 7–12 days to allow for differentiation. Under these conditions, the cells express podocyte markers including synaptopodin, nestin, and CD2AP. All cells were used between passages 17 and 25.

Human urine derived podocyte-like epithelial cells (HUPEC) carrying genes encoding a temperature-sensitive variant of the simian virus (SV40) large tumor antigen and human telomerase were cultured and maintained, as previously described (Sakairi et al. 2010 (link)). Briefly, podocytes were grown on type I collagen-coated flasks (Fisher Scientific, Rochester, NY) at 33°C (growth-permissive conditions) in growth media (RPMI 1640 medium [Sigma, St. Louis, Missouri], 10% fetal bovine serum [Hyclone, Logan, UT], 15 mmol/L HEPES, 5 mmol/L l-glutamine, 100 U/mL penicillin, 100 U/mL streptomycin, 5% CO₂ atmosphere). After reaching confluence, cells were seeded onto 35-mm type I collagen-coated dishes and maintained at 37°C (growth-restrictive conditions) for 7–12 days to allow for differentiation. All cells were used between passages 17 and 25.