Establishment and Maintenance of Human Cell Lines

Human cancer cell lines A549 and HeLa cells were purchased from RIKEN CELL BANK and cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Wako, Japan) supplemented with 1% penicillin–streptomycin and 10% fetal bovine serum (FBS; HyClone, USA) and were maintained at 37 ℃ in an atmosphere of 5% CO₂-95% air. Rho 0 A549 (ρ⁰ A549) and Rho 0 HeLa (ρ⁰ HeLa) cells were established as previously described by culturing in the presence of ethidium bromide[26 (link)] (EtBr; Sigma, USA) (50 ng/mL), 1 × sodium pyruvate solution (Wako, Japan) (110 μg/mL), and uridine (Sigma, USA) (50 μg/mL) in RPMI 1640 supplemented with 10% FBS for more than 20 passages. Control parental A549 and HeLa cells were maintained in standard culture medium for the same period. We prepared three clones of BMSCs, MSC 1, 2, and 3 using BMSCs from Lonza (Basel, Switzerland). Human BMSCs were isolated as described previously [24 (link)] and cultured in DMEM/Ham’s F-12 medium (Wako, Japan) containing 15% FBS, 1% penicillin–streptomycin, and 10 ng/mL basic fibroblast growth factor (bFGF; Abcam; USA) (in 5% CO₂, at 37 °C) until 80% confluent.