3.7. Cell Culture

U87 MG (ATCC, HTB-14™) is a human malignant glioblastoma–astrocytoma cell line that is classified as grade IV. The cells were adherent epithelial cells and were cultured in DMEM (Dulbecco’s modified Eagle medium) + GlutamaxI (Gibco, Invitrogen) supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin/streptomycin in a 5% CO₂ incubator at 37 °C under humidified atmosphere.

The cellular viability after CMBA treatment either alone or after neutron irradiation was determined using the MTT assay based on the reduction of a yellow tetrazolium salt to purple formazan by metabolic active cells, as previously described [23]. For the selection of the concentration used in the irradiation studies, adherent cells (~10⁴ cells/200 µL) were incubated with CMBA in medium at serial concentrations in the range of 6.3–200 µM for 6 h at 37 °C, 5% CO₂. Next, the medium was replaced by 200 μL of MTT (0.5 mg/mL PBS), and cells were incubated for a further 3 h. The water-insoluble formazan that was produced was then solubilized with 200 µL DMSO. The cellular viability was evaluated by measuring the absorbance at 570 nm in each well, which was taken as control (100% viable cells) for non-treated cells. A similar procedure was applied after neutron irradiation, i.e., 24 and 72 h post-irradiation.

U87 cells (~2 × 10⁶ cells/5 mL medium) were cultured in T25 flasks and incubated for 24 h to allow adherence. The medium was discarded, and CMBA was added to the cells. The compound was first diluted in DMSO and then in medium at different time points to 200, 350, and 500 μM. After 3 h of exposure at 37 °C, the solutions were removed, and cells were washed twice with cold PBS, detached with trypsin–EDTA solution (Gibco), and centrifuged to obtain a cellular pellet. The cytosol, membrane/particulate, cytoskeletal, and nuclear fractions were extracted using a FractionPREP™ cell fractionation system (BioVision, USA) according to the manufacturer’s protocol. The boron content in the different fractions was analyzed by a Thermo X-Series Quadrupole inductively coupled plasma mass spectrometer (ICP-MS) equipped with Ni cones and a glass concentric nebulizer (Meinhard, 1.0 mL min⁻¹) refrigerated with a Peltier system. After digestion of the samples in 0.5 mL nitric acid (65%) (100 °C, 12 h) in a closed pressurized microwave digestion unit (Mars5, CEM) with a medium-pressure HP500 vessel, the samples were then diluted in ultrapure water to obtain 2.0% (v/v) nitric acid. The instrument was tuned using a multielement ICP-MS 71 C standard solution (Inorganic Venture). Indium (¹¹⁵In) at 10 µg/L was used as the internal standard.

U87 cells were cultured in T25 culture flasks or in well plates according to the subsequent biological evaluation. For the radiobiological studies, cells (~10⁶/5 mL) were cultured in T25 culture flasks and incubated with CMBA at three different concentrations (50, 100, and 200 μM) 1 h prior to the irradiations. For the cytotoxic effect induced by neutron radiation, cells (approx. 10⁴) were cultured in 96-well plates and incubated with the compounds at 50, 100, and 200 μM. Then, the flasks or plates were placed into the thermal column of the Portuguese Research reactor (RPI), and cells were irradiated at room temperature for 5 h at 600 kW reactor power, corresponding to a maximum thermal neutron fluence of 5.8 × 10¹¹ n cm⁻² and photon dose imparted to the cells of 810 mGy.

U87 cells were exposed to the irradiation procedure as described above. The number of micronuclei (MN) was determined by the cytokinesis blocked micronuclei assay. Briefly, 2 mg/mL cytochalasin-B was added to the medium 20 h after irradiation to arrest cytokinesis and cells were cultured for more 24 h. Then, cells were subjected to a hypotonic treatment (RPMI medium, water, and FBS), harvested, centrifuged (800 rpm, 10 min.), re-suspended twice in the wash solution (RPMI medium, water and FBS), and centrifuged again (800 rpm, 8 min.). The cells were fixed for 20 min. in cold methanol:acetic acid (3:1). Slides were prepared and stained in a solution of 4% Giemsa dye in phosphate buffer (pH 6.8) for 8 min. The slides were coded and scored under a light microscope (Zeiss, Portugal) at 40× magnification. MN were identified according to a previously described method [28]. The frequency of binucleated (BN) cells containing one or more MN was also scored.

U87 cells were washed three times with PBS and fixed with 4% formaldehyde in PBS for 15 min. After washing with PBS, cells were permeabilized with Triton X-100 (0.5%) at room temperature for 5 min., which was followed by two washing steps with 1% BSA in PBS. Then, cells were incubated with an anti-γ-H2AX primary antibody (mouse anti-γ-H2AX (ser139), Stressgen, bioreagents Corp., Canada) at 2 μg/mL for 1 h. After being washed twice with 1% BSA in PBS, cells were incubated with a FITC-conjugated anti-mouse secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 1 mg/mL for 1 h, followed by three washing steps with PBS. After incubation with Hoechst (Sigma-Aldrich, St. Louis, MO, USA) at 1 μg/mL for 5 min, cells were mounted in anti-fade mounting media (Vectashield H-100, Vector Laboratories, Burlingame, CA, USA).