Ham s f 12 dmem f12

We isolated FLSs from the synovial tissues using the modified tissue culture method [35 (link)], shredded fresh synovial tissues into small pieces, and digested them in type I collagenase (Sigma-Aldrich, St. Louis, MO, USA) for 2 h at 37 °C. The cells were cultured with complete Dulbecco’s modified Eagle’s medium–Ham’s F-12 (DMEM/F12; Gibco Life Technologies, Shanghai, China) containing 100 g/ml streptomycin, 100 units/ml penicillin, and 20% fetal bovine serum (FBS; Gibco Life Technologies, Australia) in a humidified 5% CO₂ incubator. Our in vitro study used FLSs from passages 3–5.

The human cell line U373 (American Type Culture Collection, ATCC, Rockville, MD, USA), astrocytoma grade III/glioblastoma, was kindly provided by Professor Pierluigi Navarra, Institute of Pharmacology, Catholic University of the Sacred Heart, Rome.
The human cell line U87 (ATCC, USA), astrocytoma grade III/glioblastoma, was kindly provided by Dr. Emilio Ciusani, National Neurological Institute “Carlo Besta”, Milan [42] (link).
The human LI cell line, astrocytoma grade IV, was kindly donated by Professor Gabriella Zupi, Regina Elena Institute, Rome [43] (link), [44] (link).
The three cell lines (parental cell lines) were cultured in Dulbecco’s modified Eagle’s medium/Ham’s F-12 (DMEM/F12) (Gibco, Carlsbad, CA) containing 10% fetal bovine serum (FBS, Euroclone, Milan, Italy), antibiotics (penicillin/streptomycin 100 IU/ml/100 µm/ml, Euroclone), 4-2-hydroxyethyl-1-piperazinyl-ethanesulfonic acid (10 mM, Euroclone), glutamine (2 mM, Euroclone) and glucose (0.3% w/v, Sigma-Aldrich, St Louis, MO, USA), hereafter named: standard medium. The cells, which grow in monolayer, were subcultured to confluence, and maintained at 37°C in humidified atmosphere air: CO₂ (95%: 5%).

The human glioblastoma cell line U-87 MG and human pharynx squamous carcinoma cell line FaDu were obtained from the Korean cell line bank (KCLB; Seoul, Korea). The human tongue squamous carcinoma cell line SAS was obtained from the Japanese collection of research bioresources cell bank (JCRB; Tokyo, Japan). U-87 MG and FaDu cells were cultured in minimum essential medium (MEM) (Gibco, New York, NY, USA) with 2 mM L-glutamine (Gibco), 100 units/mL penicillin-streptomycin (Gibco), and 10% (v/v) fetal bovine serum (Gibco) at 37 °C under a 5% CO₂ atmosphere. SAS cells were cultured in Dulbecco’s modified eagle medium/Ham’s F-12 (DMEM/F12) (Gibco) with 100 units/mL penicillin-streptomycin (Gibco) and 10% (v/v) fetal bovine serum (Gibco) at 37 °C under a 5% CO₂ atmosphere.