T75 flask

To complete the evaluation of complexes C1, C3, and C4, in vitro biological behavior was assessed. Two different cell lines derived from prostatic adenocarcinoma were used, LNCaP, which express the AR, and PC3 cells, which do not express it.
The adherent cell line LNCaP (ATCC^® CRL-1740™, American Type Culture Collection, Manassas, VA, USA) was cultured in a conditioned RPMI 1640 medium (Capricorn Scientific, Ebsdorfergrund, Germany) supplemented with 10% fetal bovine serum (Capricorn Scientific), 100 U/mL of penicillin (Sigma-Aldrich, St. Louis, MI, USA), and 100 μg/mL of streptomycin (Sigma-Aldrich) in T75 flasks (Greiner Bio-one, Frickenhausen, Germany) at 37 °C and 5% CO₂. The studies were performed with monolayer cultures with less than 25 passages.
The adherent cell line PC3 (ATCC^® CRL-1435™, American Type Culture Collection, Manassas, VA, USA) was cultured in a DMEM medium (A1316, 9050 PanReac AppliChem, ITW Reagents, Darmstadt, Germany) supplemented with 10% fetal bovine serum, 100 U/mL of penicillin, and 100 μg/mL of streptomycin in T75 flasks (Greiner Bio-one, Kremsmünster, Austria) at 37 °C and 5% CO₂. The studies were performed with monolayer cultures with less than 20 passages.
Cellular uptake, efflux, and binding studies for each complex were performed.

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.

Test solutions were analysed for cytotoxicity using the Cell Titer 96 AQueous assay method (Promega, Southampton, UK) and were performed in a 96 well plate (96WP) with a known concentration of Vero cells in each well. A density of 2.5 x 10³ cells per well were used for each plate prepared, by the trypsinisation of 80% confluent Vero cells in a T75 flask (Greiner Bio-One, Stonehouse, UK) and reconstituted in fresh DMEM Cells were seeded into a 96WP and incubated for 24 h at 37°C under 5% CO₂. The medium was removed by aspiration, the cells washed three times with phosphate-buffered saline then immediately replaced with the dilutions of test materials. Cells were incubated for 6, 24, 48 and 72 h. Cell Titer 96 AQueous solution was used to detect cell viability and was added to each well and the plates incubated for 1 h at 37°C under 5% CO₂. Optical density was then determined at 492 nm, with the blank subtracted from each sample reading and the density mean for the control cells arbitrarily assigned a value of 100% (n = 3).