L929 fibroblast cell line

The human lung adenocarcinoma cell lines (A549 and H1975), the murine L929 fibroblast cell line, and the human erythroleukemia cell line K562 were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA). The human melanoma SSM2c cell line was kindly provided by Dr Barbara Stecca [56 (link)]. A549, H1975, SSM2c, and L929 cells were grown in Dulbecco’s modified Eagle medium, containing 4500 mg/L glucose (DMEM 4500, GIBCO, Thermo Fisher Scientific, Waltham, MA USA) supplemented with 10% fetal calf serum (FCS) at 37 °C in a humidified incubator containing 10% CO₂. 1.0 × 10⁶ cells were seeded in 100 mm Sarstedt dishes and propagated every 3 days by incubation with a trypsin–EDTA solution. The human erythroleukemic cell line K562 was maintained at 37 °C in RPMI-1640 medium (GIBCO, Thermo Fisher Scientific, Waltham, MA USA) supplemented with 10% FCS in T25 culture flasks (Sarstedt, Milano, Italy) in humidified incubator with 5% CO₂. For 3D co-cultures, the L929 murine fibroblast cell line was used as a proof of concept. L929 cells were grown in DMEM 4500 supplemented with 10% FCS in the same culture conditions. Cell cultures were periodically monitored for mycoplasma contamination using Chen’s fluorochrome test.