Conditionally Immortalized Proximal Tubule Cells

Conditionally immortalized proximal tubule epithelial cells (ciPTEC) were developed as described by Wilmer et al. with informed consent of the donors in accordance with the approved guidelines of the Radboud Institutional Review Board (21 (link)). Cells were seeded 7 days prior to the experiment at their corresponding density (55,000 cells/cm² for ciPTEC parent cells, 63,000 cells/cm² for ciPTEC-OAT1, and 82,000 cells/cm² for ciPTEC-OAT3) and grown for 1 day at 33°C and 5% v/v CO₂ to allow proliferation, enabled by the temperature-sensitive mutant of SV large T antigen (SV40T). Next, cells were cultured for 6 days at 37°C and 5% v/v CO₂ to stimulate differentiation and formation of an epithelial monolayer, described as “maturation.” Cells were cultured using Dulbecco’s modified eagle medium (DMEM HAM’s F12, Life Technologies, Paisly, UK), 5 μg/ml insulin, 5 μg/ml transferrin, 5 μg/ml selenium, 35 ng/ml hydrocortisone, 10 ng/ml epidermal growth factor (EGF), 40 pg/ml tri-iodothyronine (Sigma, St. Louis, USA), and 10% fetal calf serum (FCS, Greiner Bio One, Kremsmuenster, Austria). Medium was refreshed every second day, supplemented with 1% penicillin/streptomycin (pen/strep, Invitrogen, Carlsbad, USA) at 33°C and without pen/strep at the maturation temperature of 37°C. Three T3 mouse-fibroblast (3 T3) cells were cultured at 37°C and used only as irradiated non-proliferating feeder cells for sub-cloning procedures upon transduction, as described (21 (link)).