150 cm2 cell culture flask

Male C57BL/6 mice were sacrificed and both femur and tibiae were harvested and marrow was flushed out with sterile serum-free SIGMA Stemline medium (St Louis, MO) using a 28.5 gauge needle fitted to a 1 ml syringe in a 50 ml sterile tube containing 10 ml of serum-free Stemline medium. Bone marrow clumps were removed by repeated passing through a 16.5 gauge needle fitted to a 10 ml syringe until a homogeneous single cell suspension is achieved. The cells are pelleted via centrifugation at 1200 rpm, washed three times using serum-free medium and finally re-suspended in 50 ml Stemline medium containing 10% fetal bovine serum and antibiotics. The cells are further diluted with serum containing Stemline medium to 100 ml and distributed to four 150 cm² cell-culture flasks (Corning Inc. Corning, NY) for maintenance in a moist chamber with 95% O₂ and 5% CO₂ supply. After two days the culture medium was collected and centrifuged at high speed to get rid of cells and debris. The supernatants were pooled and used directly for the culture of 3-D, PC3 beads.

Isogenic SW48 colorectal carcinoma cell lines were sourced from Horizon Discovery Ltd (Cambridge, UK). All of these cell lines were generated using a recombinant Adeno-Associated Virus (rAAV)-based gene editing platform according to published methods (Arena et al. 2007 (link); Vartanian et al. 2013 (link)). All cell lines were cultured in McCoy’s 5A medium (Gibco, Life Technologies, UK), supplemented with 10% foetal bovine serum (FBS) (Gibco, UK). Cells were grown in 150 cm² cell culture flasks (Corning, UK), incubated at 37 °C in a humidified atmosphere containing 5% CO₂ and passaged routinely until they reached approximately 80–90% confluence. All experiments were performed in five biological replicates.

To obtain GBM-derived exosomes, T98G and U373 cell lines were plated in T75 cm² flasks. After 70% confluency in 150 cm² cell culture flasks (Corning, USA), 25 mL of complete medium was added. Subsequently, the Total Exosome Isolation Reagent (Invitrogen™—Cat. 4478359, Waltham, MA, USA) protocol for the effective isolation of exosomes was used as previously described [22 (link),23 (link)]. Briefly, conditioned medium and reagent (2:1) were mixed and incubated at 2 °C to 8 °C overnight. After incubation, the samples were centrifuged at 10,000× g for 1 h at 2 °C to 8 °C in order to pull down the exosomes at the bottom of the tube.
A wide range of exosome concentrations was used as follows: 1–5–10–50–100 µg/mL. Such concentrations were selected on the basis of similar studies reported in literature [24 (link),25 (link),26 (link),27 (link)]. In detail, exosomes at different concentrations were added into 96 well plates, containing primary neuron cells at 85% confluence to assess the toxicity of GBM-derived exosomes. After 24 h of incubation (at 37 °C, 5% CO₂ and 95% humidity), MTT assay and immunohistochemical staining were performed and TAS, TOS, GSH and LDH levels were evaluated. Untreated cells were used as controls for biochemical and immunofluorescence evaluations.