GSCs were cultured in a selective medium for NSC, composed of DMEM F-12 and Neurobasal 1:1, B-27 supplement without vitamin A (Life Technologies Italia, Milan, Italy), 2 mM L-glutamine (Euroclone S.p.A., Milan, Italy), 10 ng/mL recombinant human bFGF and 20 ng/mL recombinant human EGF (Miltenyi Biotec, Bergish Gladbach, Germany), 20 UI/mL penicillin and 20 µg/mL streptomycin (Euroclone S.p.A., Milan, Italy). After isolation, the medium was replaced every 3 days to remove stroma and red blood cells residues, catabolic products and to supply fresh nutrients. Debris and adherent death cells generally were eliminated after a couple of passages. The isolated cells propagate in culture as free-floating spheres defined as tumorspheres [6 (link)], which appeared in 15–20 days of culture after isolation. When tumorspheres reached an average size of 100 μm in diameter, the culture was ready to be passed and expanded. At each passage (P), tumorspheres were mechanically dissociated using a sterilized p200 pipette set at 180–200 µL and pipetting up and down 100–150 times to achieve a single-cell suspension.
Recombinant human bfgf
Manufactured by Miltenyi Biotec
Sourced in Italy, United States, Germany
Recombinant human bFGF is a protein that plays a key role in cellular processes. It is a member of the fibroblast growth factor family and is involved in the regulation of cell growth, differentiation, and tissue repair.
Automatically generated - may contain errors
Lab products found in correlation
Recombinant human egf, by Miltenyi Biotec (7 mentions)
Penicillin, by Euroclone (6 mentions)
Streptomycin, by Euroclone (6 mentions)
Laminin, by Thermo Fisher Scientific (5 mentions)
B 27 supplement without vitamin a, by Thermo Fisher Scientific (5 mentions)
L glutamine, by Euroclone (3 mentions)
Heparin, by STEMCELL (2 mentions)
Dmem f12, by Euroclone (2 mentions)
Neurobasal 1 1, by Thermo Fisher Scientific (2 mentions)
Neurobasal 1, by Thermo Fisher Scientific (2 mentions)
Dmem f12, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Human recombinant egf, by STEMCELL (1 mentions)
Rlt lysis buffer, by Qiagen (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Neurocult ns a proliferation medium, by STEMCELL (1 mentions)
Celigo s imaging cytometer, by Revvity (1 mentions)
Valproic acid, by Merck Group (1 mentions)
Temozolomide, by Merck Group (1 mentions)
8 protocols using recombinant human bfgf
1
Isolation and Expansion of Glioblastoma Stem Cells
2
Sphere Culture Protocols for Cell Expansion
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For sphere cultures, cells were seeded at a low density (3000 viable cells/cm2) onto non-adherent plates and cultured in serum-free DMEM/F-12 medium, supplemented with 2% B27 (Gibco Life Technologies, Rockville, MD, USA), 20 ng/mL recombinant human bFGF (Miltenyi Biotec, Bergisch Gladbach, Germany), 20 ng/mL recombinant human EGF (StemCell Technologies, Vancouver, BC, Canada), 0.0002% heparin (StemCell Technologies, Vancouver, BC, Canada) and antibiotics (100 U/mL penicillin, 100 µg/mL streptomycin, Gibco Life Technologies, Rockville, MD, USA). Every 3 days, 25% of the medium was replaced. After 7–14 days of culturing, the spheres were collected by centrifugation at 110× g and lysed in Qiagen RLT lysis buffer for RNA isolation or lysed in buffer supplemented with complete protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN, USA) for blotting.
3
SRH Sphere Formation Assay Protocol
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SRH single cell suspensions that were generated by trypsin-EDTA dissociation were prepared for sphere formation assays. The cells were seeded in NeuroCult™ NS-A Proliferation Medium (Stemcell Technologies, Vancouver, BC, Canada), supplemented with 20 ng/mL recombinant human EGF (Miltenyi Biotech Bergisch Gladbach, Germany), 10 ng/mL recombinant human bFGF (Miltenyi Biotech Bergisch Gladbach, Germany), and 2 µg/mL heparin (Stemcell Technologies, Vancouver, BC, Canada) at a density of 10–1000 cells/well in Nunclon Sphera™ ultra-low attachment flat bottom 96-well plates (Thermo Fisher Scientific, Waltham, MA, USA). SRH spheres were cultured at 37 °C in humidified air containing 5% CO2 for seven days before being scored with the Celigo® S Imaging Cytometer (Nexcelom Bioscience, Lawrence, MA, USA). The sphere forming efficiency (%) was calculated as the number of tumor spheres divided by number of cells seeded × 100.
4
Glioma Stem Cell Lines Expansion
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All the glioma stem cell (GSC) lines used in this work (GBM2, GBM7, G144, G179, G166, GliNS2, GBM04) were isolated from patients affected by glioblastoma and extensively characterized for their stemness properties. GBM2, GBM7, G144, G166, GliNS2, GBM04 derived from classic glioblastoma multiforme, while G179 derived from a giant cell variant glioblastoma. All the GSC lines have been already expanded in vitro as stable cell lines and used as powerful model for studying their biology and testing drug susceptibility [26 (link), 27 (link)]; furthermore their cytogenomic and epigenomic profiles were well characterized [28 (link)].
The stemness properties of these GSC lines were periodically monitored, as already described [29 (link)]. Cell expansion was carried out in a proliferation permissive medium composed by DMEM F-12 (Euroclone) and Neurobasal 1:1 (Invitrogen), B-27 supplement without vitamin A (Invitrogen), 2 mM L-glutamine (Euroclone), 10 ng/ml recombinant human bFGF and 20 ng/ml recombinant human EGF (Miltenyi Biotec), 20 UI/ml penicillin and 20 μg/ml streptomycin (Euroclone) (complete medium). GSCs were cultured in adherent culture condition in T-25 cm3 flasks coated with 10 μg/ml laminin (Invitrogen), in 5% CO2/95% O2 atmosphere.
The stemness properties of these GSC lines were periodically monitored, as already described [29 (link)]. Cell expansion was carried out in a proliferation permissive medium composed by DMEM F-12 (Euroclone) and Neurobasal 1:1 (Invitrogen), B-27 supplement without vitamin A (Invitrogen), 2 mM L-glutamine (Euroclone), 10 ng/ml recombinant human bFGF and 20 ng/ml recombinant human EGF (Miltenyi Biotec), 20 UI/ml penicillin and 20 μg/ml streptomycin (Euroclone) (complete medium). GSCs were cultured in adherent culture condition in T-25 cm3 flasks coated with 10 μg/ml laminin (Invitrogen), in 5% CO2/95% O2 atmosphere.
5
Characterization of Glioblastoma Stem Cell Lines
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All the GSC lines used in this work (GBM2, G144, G179, G166, GliNS2, and GBM04) were isolated from patients affected by GBM and extensively characterized for their stem cell properties. GBM2, GBM7, G144, G166, GliNS2, and GBM04 derived from classic glioblastoma multiforme, while G179 derived from a giant cell variant glioblastoma. All the GSC lines have been already expanded in vitro as stable cell lines and used as powerful model for studying their biology and testing drug susceptibility [18 (link), 19 (link)]. In 2013, our research group characterized their cytogenomic and epigenomic profiles [20 (link)]. The stemness properties of the GSC lines were periodically monitored, as already described in Baronchelli et al. 2013 [20 (link)]. Cell expansion was carried out in a proliferation permissive medium composed by DMEM F-12 (Euroclone) and Neurobasal 1 : 1 (Invitrogen), B-27 supplement without vitamin A (Invitrogen), 2 mM L-glutamine (Euroclone), 10 ng/mL recombinant human bFGF and 20 ng/mL recombinant human EGF (Miltenyi Biotec), and 20 UI/mL penicillin and 20 μg/mL streptomycin (Euroclone) (complete medium). GSCs were cultured in adherent culture condition in T-25 cm2 flasks coated with 10 μg/mL laminin (Invitrogen), in 5% CO2/95% O2 atmosphere.
6
Glioblastoma Stem Cell Culture Protocol
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The six GSC lines used in this work (GBM2, GBM7, G144, G166, G179 and GliNS2) have been isolated from patients affected by GBM [20] (link), [45] (link) and in 2013, our research group characterized their cytogenomic and epigenomic profiles [2] (link). The stemness properties of the GSC lines were periodically monitored, as already described [2] (link). Cells were cultured in adherent culture condition using 10 μg/ml laminin (Invitrogen) in a proliferation permissive medium composed by DMEM F-12 and Neurobasal 1:1 (Invitrogen), B-27 supplement without vitamin A (Invitrogen), 2 mM L-glutamine, 10 ng/ml recombinant human bFGF and 20 ng/ml recombinant human EGF (Miltenyi Biotec), 20UI/ml penicillin and 20 μg/ml streptomycin (Euroclone) (complete medium).
7
Glioblastoma Stem Cell Characterization
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The GSC lines (GBM2, G144, G166, GBM04) were isolated from patients affected by glioblastoma and characterized for their stemness properties from a genetic and molecular point of view (Baronchelli et al., 2013 (link)). All the GSC lines were already expanded in vitro as stable cell lines and used as powerful model for studying their biology and testing drug susceptibility, furthermore their cytogenomic and epigenomic profiles were well characterized (Riva et al., 2014 (link), 2018 (link); Cilibrasi et al., 2017 (link)). GSC were cultured in adherent culture condition using 10 mg/ml laminin (Invitrogen) in a proliferation permissive medium composed by DMEM F-12 and Neurobasal 1:1 (Invitrogen), B-27 supplement without vitamin A (Invitrogen), 2 mM L-glutamine, 10 ng/ml recombinant human bFGF and 20 ng/ml recombinant human EGF (Miltenyi Biotec), 20 UI/ml penicillin and 20 g/ml streptomycin (Euroclone; complete medium).
8
Glioblastoma Stem Cell Culture and Drug Treatments
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Cell lines and cell culture conditions. The seven GSC lines used in this study (GBM2, GBM7, GBM04, G144, G166, G179 and GliNS2) were isolated from patients affected by GBM (22, 23) and, in 2013, our research group characterized their cytogenomic and epigenomic profiles (24) . Cells were cultured in neural stem cell-specific medium, and their stem-like properties were periodically monitored, as previously described (22) (23) (24) . Cells were cultured in an adherent culture condition using 10 µg/ml laminin (Invitrogen) in a proliferation permissive medium composed of Dulbecco's modified Eagle's medium (DMEM)/F-12 and Neurobasal 1:1 (Invitrogen), B-27 supplement without vitamin A (Invitrogen), 2 mM L-glutamine, 10 ng/ml recombinant human bFGF and 20 ng/ml recombinant human EGF (Miltenyi Biotec), 20 uI/ ml penicillin and 20 µg/ml streptomycin (Euroclone).
Drug treatments. Valproic acid (sodium salt; Sigma) was dissolved in sterile water to a stock concentration of 0.5 M. Temozolomide (Sigma) was dissolved in sterile DMSO at the final concentration of 0.05 M. All drugs were stored at -20˚C.
In vitro treatments were performed using 2 mM VPA for 24, 48,72, 96 h and 14 and 30 days; with regard to TMZ, we administered 50, 100, 200 or 400 µM TMZ for 48 or 72 h.
Drug treatments. Valproic acid (sodium salt; Sigma) was dissolved in sterile water to a stock concentration of 0.5 M. Temozolomide (Sigma) was dissolved in sterile DMSO at the final concentration of 0.05 M. All drugs were stored at -20˚C.
In vitro treatments were performed using 2 mM VPA for 24, 48,72, 96 h and 14 and 30 days; with regard to TMZ, we administered 50, 100, 200 or 400 µM TMZ for 48 or 72 h.
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