Glioma Cell Line Cultivation and Characterization

Materials for tissue culture were purchased either from Euroclone Italia (Euroclone S.p.A, Milan, Italy) or from ATCC (Manassas, VA, USA). Three human glioma cell lines, U251(EATCC; 09063001), U87MG (ATCC; HTB-14), and T98G (ATCC; CRL190), were cultured at 37°C in 5% CO₂ in Dulbecco’s modified Eagle medium (DMEM) containing 10% (v/v) fetal bovine serum, 4 mM glutamine, 100 IU/ml penicillin, 100 μg/ml streptomycin, and 1% nonessential amino acids (Thermo Fisher Scientific Inc., Carlsbad, CA, USA). The risk of working with misidentified and/or contaminated cell lines was minimized by using GBM cells at very low passages and periodic short tandem repeat (STR) DNA profiling. Luciferase-tagged U87MG (U87MG-Luc) cells were generated and provided by Jari E. Heikkila (Abo Akademi University, Turku, Finland). A GBM patient-derived stem cell (GSC) lines (BT48EF and CSCs-5) were provided by J. Gregory Cairncross and Samuel Weiss (University of Calgary, Canada) and by M. Izquierdo (Universidad Autónoma de Madrid, Spain), respectively (Luchman et al., 2012 (link); Mendiburu-Elicabe et al., 2014 (link)). Isolated neurospheres of U87 and GSC cells were assayed for ‘stemness’ properties in terms of clonogenic capacity and positivity for established stem cell markers (Gravina et al., 2019 (link)).