Joklik modified eagle s medium

After tumescent liposuction, lipoaspirates were centrifuged at 3×10³g for 3 minutes and the stromal vascular fraction was collected in a sterile container. The samples were enzymatically dissociated in a 0.05% Collagenase type II solution (Sigma-Aldrich) in Joklik modified Eagle’s Medium (Sigma-Aldrich) for 20 minutes at 37°C. Collagenase activity was stopped by the addition of 0.1% BSA (Sigma-Aldrich) solution in Joklik modified Eagle’s Medium (Sigma-Aldrich). Cell suspension was centrifuged at 1×10³g for 10 minutes. Samples collected from different subjects were kept distinct and used to obtain distinct hAT-MASC cell lines.
2.0×10⁶ freshly isolated human cells were plated onto 100 mm human fibronectin (Sigma-Aldrich) coated dishes (BD Falcon) in an expansion medium composed as follows: 60% low glucose DMEM (Invitrogen), 40% MCDB-201, 1 mg/mL linoleic acid-BSA, 10⁻⁹ M dexamethasone, 10⁻⁴ M ascorbic acid-2 phosphate, 1X insulin-transferrin-sodium selenite (all from Sigma-Aldrich), 2% fetal bovine serum (StemCell Technologies), 10 ng/ml human or murine PDGF-BB, 10 ng/ml human or murine EGF (both from Peprotech EC). Medium was replaced with fresh one every 4 days. Once cells reached 70–80% of confluence, they were detached with 0.25% trypsin-EDTA (Sigma-Aldrich) and re-plated at a density of 1–2×10³/cm².

Glioma-associated stem cells (GASC) were isolated from the three regions, displaying a progressive intensity of fluorescence for 5-aminolevulinc acid and maintained, in vitro, applying, with minor modifications, a protocol optimized for culturing multipotent adult stem cells from normal and neoplastic human tissues. Briefly, GBM fragments were first mechanically disaggregated with scalpels and then enzymatically dissociated, in a 0.025% Collagenase type II solution (Worthington) in Joklik modified Eagle’s Medium (Sigma-Aldrich, St.Louis, MO, USA) for 5 min at 37 °C. Collagenase activity was stopped by adding 10% Fetal Bovine Serum in Joklik modified Eagle’s Medium (Sigma-Aldrich). Cell suspension was centrifuged at 500× g for 10 min after being filtered through a sieve (BD Falcon, Franklin Lakes, NJ, USA) in order to select a population less than 40 μm in diameter. 2.0 × 10⁶ freshly isolated human cells were plated onto 100 mm diameter, human fibronectin (Sigma-Aldrich)-coated dishes (BD Falcon), in an expansion medium composed as follows: 60% low glucose DMEM (Invitrogen), 40% MCDB-201, 1 mg/mL linoleic acid-BSA, 10⁻⁹ M dexamethasone, 10⁻⁴ M ascorbic acid-2 phosphate, 1× insulin-transferrin-sodium selenite (all from Sigma-Aldrich), 2% foetal bovine serum (StemCell Technologies, Cambridge, UK), 10 ng/mL human PDGF-BB, 10 ng/mL human EGF (both from Peprotech EC, London, UK).