Pericyte growth medium

Placental pericytes (Pl-PCs; PromoCell) and bone marrow derived mesenchymal stromal cells (BM-MSCs; PromoCell) were thawed and seeded into untreated/uncoated flasks. Pl-PCs were seeded at a density of 3–4 × 10⁴ cells per cm² into 1 T-150 flask. Pl-PCs were cultured and expanded in Pericyte Growth Medium (PromoCell). BM-MSCs were seeded at a density of 4 × 10⁴ cells per cm² into 1 T-150 flask. BM-MSCs were cultured and expanded in Mesenchymal Stem Cell Growth Medium (PromoCell). Both Pl-PC and BM-MSC cultures were fed once every 2–3 days and cells passaged at 80–90% confluency.

All fresh glioblastoma tumor samples were initially washed twice with 1x HBSS and centrifuged (80 xg, 90 s). Following transfer onto 10 cm culture dishes, specimens were cut into fragments <1 mm³ and part of material was further enzymatically dispersed (37 °C, 45 min – 1.5 h, depending on the fragment size) with collagenase type IV (200 U/mL, Sigma-Aldrich), dispase (1 U/mL; STEMCELL Technologies) and DNaseI (3 μg/mL; STEMCELL Technologies). Following filtration through 70 μm cell strainer cells were centrifuged (80 xg, 90 s) and subsequently suspended in Astrocyte Growth Medium (AGM; Lonza) for adherent GB cultures or Pericyte Growth Medium (PromoCell) for pericyte cultures. Depending on proliferation rate, cells were passaged with StemPro Accutase (Gibco) to a new culture dish every 7–14 days. For further immunofluorescence analyses, cells were passaged onto 4 well plates with coverslips and left to adhere.

Endothelial cell basal medium and pericyte growth medium were purchased from PromoCell (Heidelberg, Germany). Dulbecco’s modified Eagle’s medium (DMEM) low glucose was from Lonza (Cologne, Germany). Human TNF-α was from Provitro (Berlin, Germany). MA was purchased from Santa Cruz Inc. (Heidelberg, Germany). Fluorescein isothiocyanate-labeled dextran 150,000, rhodamine 6 G and peroxidase-labeled-streptavidin were purchased from Sigma-Aldrich (Munich, Germany). Mayer’s hemalaun solution was from Merck (Darmstadt, Germany). 3-Amino-9-ethylcarbazole was obtained from Abcam (Cambridge, UK). Ketamine (Ursotamin) was from Pharmacia GmbH (Erlangen, Germany) and xylazine (Rompun) was from Bayer (Leverkusen, Germany).

After CD34+ cell population expansion, the cells were split into two halves and plated in Endothelial Cell Growth Medium MV2 and Pericyte Growth Medium (Promocell, Heidelberg, Germany) at a concentration of 1 x 10⁶ cells/2ml/9.6cm² in fibronectin-coated tissue culture dishes.

Dulbecco phosphate buffered saline (DPBS), phosphate buffered saline (PBS), human endothelial serum free medium (hESFM), a CellROX™ Deep Red Flow Cytometry Assay Kit, RNaseA/T1, TRIzol reagent and the GeltrexTM LDEV-Free Reduced Growth Factor Basement Membrane Matrix were purchased from Thermo Fisher Scientific (Thermo Fisher Scientific, Waltham, MA, USA). Propidium iodide (PI) was purchased from Miltenyi Biotec (Miltenyi Biotec, Bergisch Gladbach, Germany). RNA isolation was performed with the NucleoSpin RNA II kit (Macherey-Nagel GmbH & Co. KG, Düren, Germany). iScript cDNA synthesis kit, iTaq Universal SYBR Green Supermix and iTaq Universal Probe Supermix were used for the cDNA synthesis and qPCR analysis, all these products are by Bio-Rad (Bio-Rad Laboratories Inc., Hercules, CA, USA). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, USA). Pericyte growth medium (PGM) was purchased from PromoCell (PromoCell GmbH, Heidelberg, Germany). Sodium hydrogen sulfide (NaHS) was purchased from Cayman (Cayman Chemical, Ann Arbor, MI, USA). All plastic supports for cell culture were purchased from Corning-Becton, Dickinson (Corning-Becton, Dickinson and Company Becton Drive, Franklin Lakes, NJ, USA).