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.