Hacemos pulseras

3.2.1 Derivation and propagation of patient-derived cell lines

Primary tumour samples were placed in specimen tubes and immediately placed on ice for transport to the human tissue fume hood. Under the fume hood, tumour fragments were minced with scalpels separately, transferred to a 50mL Falcon tube, and incubated at 37.5o_C

and 5% CO2 for 45-90 minutes with 2-5 mL of Accutase (Gibco), depending on the size of the

tumour sample, to degrade extracellular material. After incubation, HBSS (without Calcium and Magnesium, Gibco) was added to the suspension for dilution. The single cell suspension was then filtered through a 40 µm cell strainer to remove debris, endothelial/vascular cells, and extracellular material. The resulting single cell suspension was spun down at 1200 g for 5 minutes. The supernatant was carefully discarded and 1-3.5 mL red blood cell lysis buffer was added to resuspend the pellet depending on the size and suspected level of blood content of the

pellet. After 10 minutes of incubation at room temperature, cells were spun down again at 1200 g for 5 minutes. The supernatant was carefully discarded and then 10 mL of HBSS was added to resuspend and wash the pellet. Washing occurred 1-3 more times depending on the size of the pellet. After a final wash and spin, the supernatant was discarded and the pellet was resuspended in serum free medium (SFM; Neurobasal A (Invitrogen, UK) with 20 mM L- glutamine, 1% PSF solution, 20 ng/ml hEGF (Sigma, UK), 20 ng/ml hFGF (R&D systems, UK), 2% B27 (Invitrogen, UK) and 1% N2 (Invitrogen, UK)). Cells were counted with a hemocytometer and then plated with SFM at a density of approximately 100,000 cells per mL in 25 or 75 cm2 _{cell culture flasks and placed in a 37.5}o_{C, 5% CO2 incubator.}

After 7-10 days, cells began to form floating neurospheres. Once there were enough neurospheres of sufficient size to be viable, neurospheres were removed from flasks, spun down at 1200 g for 5 minutes, washed with HBSS, resuspended in SFM, and added to cell culture flasks that had been pre-coated with extracellular matrix (ECM; Sigma). Neurospheres then plated down forming an adherent monolayer of glioma cells that were maintained and utilised for future experiments. To passage adherent monolayer cultures, media was removed from flask and 2-4 mL of Accutase was added for 10 minutes in 37.5o _{C incubator. Around 4}

mL of SFM was added to floating cells and suspension transferred to 15 ml Falcon tube. If needed, Accutase step was repeated to remove remaining cells from flask. Cells were then spun at 1200 g for 5 minutes, resuspended in HBSS and spun again. The supernatant was discarded and SFM was added. Cells could then be counted, re-plated, frozen down, or utilised as needed.

3.2.2 Freezing and thawing patient-derived cell lines

Depending on the growth rate of individual, patient-derived cell lines, lines were passaged every one to two weeks. Typically, with each passage, cells were counted using a hemocytometer and 500,000-1 million cells in SFM were added to freezing media (SFM with 10% DMSO) to a total volume of 1 mL. Lines were then frozen down in isopropanol freezing containers at -80o_{C before being transferred to liquid nitrogen for permanent storage.}

Individual patient-derived cell lines were thawed and re-established when necessary. Cells were removed from -80o_{C freezer or liquid nitrogen storage and placed on dry ice. 15}

in gloved hands. Once thawed, cells were quickly transferred to SFM and spun down. Supernatant was discarded and cells were washed with SFM or HBSS 1-3 more times, depending on the size of the initial pellet. Finally, 10 mL of SFM was added to pellet and solution was placed in a 75 cm2 _{culture flask and placed in a 37.5}o_{C, 5% CO2 incubator. A few}

days later, cells were transferred to ECM-coated flasks.

3.2.3 Glioblastoma stem-like cells and other glioblastoma lines

GSC lines NSC11, NSC20, and NSC23 were isolated from three human GB surgical specimens and were kindly provided by Dr. Frederick Lang (MD Anderson Cancer Center). They were maintained as neurospheres in stem cell medium composed of DMEM/F-12 (Invitrogen), B27 supplement (Invitrogen), and human recombinant bFGF and EGF (50ng/ml each, R&D Systems) at 37o_{C, 5% CO2, and5% O2, as previously reported (267,268). FACS}

was utilised to sort for CD133+ cells from each culture. To prepare cells, TrypLe Express (Sigma) was utilised to generate single cell suspensions from neurospheres. After 30 seconds of exposure, defined trypsin inhibitor (Sigma) was added and neurospheres were disaggregated. After washing, single cell suspensions were labelled with anti-CD133 antibody (PE- conjugated, Miltenyi) and FACS sorted under sterile conditions. CD133+ GSCs were expanded and utilised for all experiments. The U251 human GB cell line was obtained from the Division of Cancer Treatment and Diagnosis Tumour Repository (DCTD), National Cancer Institute (NCI), grown in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% FBS (Invitrogen), and maintained as adherent cultures at 37o_{C, 5% CO2.}

GSC lines and U251 cells were cultured less than 2 months after resuscitation. Cell lines tested negative for mycoplasma contamination by PCR analysis (Idexx BioAnalytics). All lines were transduced with lentivirus (LVpFUGQ-UbC-ffLuc2-eGFP2, provided by Viral Technology Laboratory, NCI Frederick) as single cells at a MOI of 1 to express both GFP and luciferase. GFP expression was monitored in vitro and, in some cases, cells were sorted for GFP positivity by FACS prior to in vivo implantation. For use in an in vitro experiment, GSC neurospheres were disaggregated into single cells, as described above, and seeded onto poly- L-ornithine (PO, Invitrogen)/laminin (Sigma-Aldrich) coated tissue culture dishes in stem cell media. Under these conditions, single-cell GSCs attach and grow as an adherent monolayer maintaining their CD133 expression and stem-like characteristics (269). Radiation was

delivered to in vitro cultures using a 320 kV Xray machine (Precision X-Ray Inc.) at a dose rate of 2.3Gy/min.