Materials and methods
Urothelial cells procured from porcine bladders were isolated and cultured according an adapted protocol as described by Fraser.[12] The cells were cultured in Primaria T75 flasks in keratinocyte serum-free medium (K-SFM) (Sigma Aldrich). The K-SFM was supplemented with 0.05 mg/mL bovine pituitary extract, 0.005 µg/mL recombinant epidermal growth factor (Gibco, Life Technologies), penicillin/streptomycin 1% (vol/vol) and 30 ng/mL cholera toxin. At the third passage the cells were seeded on slides for immunohistochemical staining and on inserts (360.000 cells/cm2 , transwell Costar 24-wells plate; diameter 6.5mm; pore size 0.4 µm) with a porous PET membrane for functional barrier measurements. When confluency was reached, the medium was supplied with 5% fetal calf serum (FCS) and 2mM calcium chloride (Ca2+Cl) to induce terminal differentiation. [8] Terminal differentiation was confirmed with TEER measurements and immunofluorescence analyses.