Endothelial Cell Culture and MMC Treatment

Commercially available HCAECs (cat. No. 300K-05a, Cell Applications Inc., San Diego, CA, USA) and HITAECs (cat. No. 308K-05a, Cell Applications Inc., San Diego, CA, USA) cryopreserved at the 2nd passage were used in the present research. All manipulations with cells were performed as described previously [27 (link)]. Briefly, the cells were seeded into fibronectin-coated T-75 flasks (Greiner Bio-One GmbH., Kremsmünster, Austria) containing 15 mL of a Human MesoEndo Cell Growth Medium (Cell Applications Inc., San Diego, CA, USA) and incubated in a humidified atmosphere with 5% CO₂ at 37 °C. After 3 passages, the cells were reseeded into new T-75 flasks (Greiner Bio-One GmbH., Kremsmünster, Austria) and refed (after reaching 80% confluency) with another 15 mL of a Human MesoEndo Cell Growth Medium (Cell Applications Inc., San Diego, CA, USA) containing 500 ng/mL of MMC (AppliChem, Barcelona, Spain, CAS No. 50-07-7) (treatment group) or 0.9% NaCl (control group) followed by 6 h of incubation. Then, cells were washed twice using ice-cold phosphate-buffered saline (PBS) and refed with another 15 mL of an additive-free Human MesoEndo Cell Growth Medium (Cell Applications Inc., San Diego, CA, USA) followed by 24 h of incubation. To avoid any possible batch-effects, all manipulations with HCAECs and HITAECs were performed in parallel.