Normal human astrocytes nhas

The immortalized human brain EC line hCMEC/D3 was kindly provided by Dr.Couraud from the Institut Cochin, Paris, France. ECs were developed on culture inserts (0.4-mm pore size; Corning, Lowell, MA, USA) coated with 150 μg/mL Cultrex Rat Collagen I (R&D Systems, Minneapolis, MN, USA). ECs were cultured in endothelial basal medium (EBM-2) (Lonza, Walkersville, MD, USA), containing 5% fetal bovine serum (FBS) “Gold” (PAA Laboratories, Pasching, Austria), 1% penicillin-streptomycin (Life Technologies, Paisley, UK), 1.4 μmol/L hydrocortisone (Sigma-Aldrich, St Louis, MO, USA), 1% chemically defined lipid concentrate (Life Technologies, Paisley, UK), 5 μg/mL ascorbic acid (Sigma-Aldrich), 10 mmol/L HEPES (PAA Laboratories), and 1 ng/mL human basic fibroblast growth factor (bFGF) (Sigma-Aldrich). ECs algebra were maintained at 30–40 generations. Human glioma cell line U87 and human embryonic kidney cell line 293T (HEK293T) were purchased from Shanghai Institutes for Biological Sciences Cell Resource Center and developed in Dulbecco’s modified Eagle’s medium (DMEM) of high glucose with 10% FBS (GIBCO, Carlsbad, CA, USA). Normal human astrocytes (NHAs) were purchased from Sciencell Research Laboratories (Carlsbad, CA, USA) and developed in RPMI-1640 culture medium (GIBCO, Grand Island, NY, USA) with 10% FBS. All cells were maintained at 37 °C, 5% CO₂, in a humidified incubator.

In this study, three human GBM cell lines were used: T98G (ATCC, Manassas, VA, USA), U87MG (Sigma-Aldrich, St. Louis, MO, USA) and LN229 (kindly provided by Dr. Rafał Krętowski, Department of Pharmaceutical Biochemistry, Medical University of Białystok, Poland). T98G cells were grown in MEME (Sigma-Aldrich) containing 10% fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific, Grand Island, NY, USA) and non-essential amino acids (Gibco). U87MG cells were cultured in EMEM (ATCC) supplemented with 15% FBS. LN229 cells were maintained in DMEM (Gibco) containing glucose and 10% FBS.
To establish patient-derived cultures, tumor specimens were mechanically and enzymatically dissociated and filtered through 100 μm cell strainers. Cells were resuspended in DMEM/HAM’s F12 medium (Gibco) supplemented with either 10% FBS for adherent cultures, or B-27 (1:50 dilution), epidermal growth factor (20 ng/ mL), and basic fibroblast growth factor (10 ng/mL) for sphere cultures. All the above supplements were obtained from Gibco.
Normal human astrocytes (NHAs) (ScienCell Research Laboratories, Carlsbad, CA, USA) were cultured in the astrocyte medium as recommended by the manufacturer.
All media were supplemented with penicillin and streptomycin purchased from either Gibco or ScienCell Research Laboratories. The cells were kept at 37 °C in a humidified atmosphere of 5% CO₂ and 95% air.

Five human GBM cell lines (U87, LN229, A172, T98, U251) and human embryonic kidney (HEK) 293 T cells were purchased from the Chinese Academy of Sciences Cell Bank (Shanghai, China), N3 primary culture cell were donated from Tian Tan Hospital and four drug-related cell lines (Pri GBM, N3S, Rec GBM, N3T3rd) were used from our previous study [13 (link)]. All GBM cells were maintained in high glucose DMEM medium supplemented with antibiotics (100 units/mL penicillin and 100 mg/mL streptomycin) and 10% fetal bovine serum (FBS). Normal human astrocytes (NHAs) were acquired from Sciencell Research Laboratories (Carlsbad, CA, USA) and cultured in astrocyte medium (Carlsbad, CA, USA). All cells were grown at 37 °C with 5% CO₂.