75 cm2 flask

Six human cancer cell lines, T24, Panc-1, Capan-1, DU145, HGC-27, and BGC-823, were originally purchased from the American Type Culture Collection and cultured in DMEM or RPMI1640 medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin solutions (Gibco, USA) at 37°C in a 5% CO₂ and 95% humidified atmosphere and harvested with trypsin-EDTA. Logarithmically growing cells were used for all experiments. The cells were cultivated in 75-cm² flasks (Costar, Cambridge, MA) until they reached approximately 50 to 70% confluence. The cells were then treated with varying doses of the indicated chemicals. DMSO alone was used as the vehicle control.

The human retinal pigment epithelial cell line ARPE-19 and the mouse fibroblast L929 cell lines were obtained from the American Tissue Culture Collection (ATCC, VA, USA). After thawing, the ARPE-19 cells were grown in Dulbecco’s Modified Eagle Medium and Ham’s F12 Nutrient Mixture (DMEM/F12, Gibco®, Invitrogen, Paisley, UK), and the L929 cells were grown in DMEM cell culture medium (Gibco®, Invitrogen). Both cell culture media were supplemented with 10% foetal bovine serum (FBS; Gibco®) and 1% antibiotic and antimycotic (100 U/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B; Gibco®). These cell cultures were maintained in 75 cm² flasks (Costar, Corning Inc., Corning, NY, USA) in standard culture conditions of 37 °C and 5% CO₂, changing the media every 2 days. The cells were harvested by trypsinization using 0.05% Trypsin-EDTA solution (Gibco®) at 80–90% culture confluence and further sub cultivated into culture flasks^{8 (link),9 (link)}.

After removing the umbilical arteries and vein, the Wharton Jelly tissue was cut with scissors into small pieces (1–3 mm³), placed into 6-well plates (Costar, Corning Life Sciences, Canton, MA, USA) and cultured with growth media in a humidified atmosphere with 5% CO₂ at 37 °C. Upon reaching the sufficient number of adherent cells in the 6-well plates, cells were detached using 0.25% trypsin EDTA solution (Gibco), washed with PBS 1× and re-plated into the 75 cm² flasks (Costar) with the appropriate culture medium. On reaching 80% of confluency, the cells were trypsinized, washed and resuspended into the 175 cm² flasks. The same procedure was repeated until the cells reached passage 5 (P5) of culture. The growth media that were used for the expansion of Wharton’s Jelly-Mesenchymal Stromal Cells (WJ-MSCs) was α-Minimum Essentials Medium (α-MEM, Gibco) supplemented either with 15% Foetal Bovine Serum (FBS, Gibco) or 10% PB-PL or 10% CB-PL. Each growth medium was supplemented with 10 U/mL penicillin (Gibco) and 10 μg/mL streptomycin (Gibco) and 2 mM l-glutamine (Gibco). The growth medium was changed twice every week and the cultures were maintained in a humidified atmosphere with 5% CO₂ at 37 °C.