Volatolomic Analysis of Extracellular Vesicles Extracted from Cultured Cells (GC-IMS)

Cells and Cell Culture Human epithelial cells (EPI), human hepatocellular cancer cells (Hep-G2) and human embryotic kidney cells (HEK-293) were purchased from ATCC, Teddington, UK (PCS-300-010, HB-8065 and CRL-1573.3, respectively). The cells were cultured in T175 cell culture treated flasks with filter caps (Thermo Fisher Scientific, Loughborough, UK) in the F-12 Dulbecco's modified eagle medium/nutrient mixture (DMEM-F12, Thermo Fisher Scientific Inc., Santa Clara, CA, USA), supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin (P/S), and 0.01% amphotericin B. The cultures were maintained in a humidified incubator at 38.8 °C with 5% CO2 until they reached 70-85% confluence, upon which the medium was replaced with serum-depleted DMEM-F12 to deplete the cell culture media from vesicles present in serum. EV Isolation and Characterisation The conditioned media of the three cell lines (EPI, Hep-G2 and HEK-293) were subjected to differential centrifugation. The samples were centrifuged at 300×g for 10 min to remove the potential presence of whole cells, then the supernatant was centrifuged at 2,000×g for 10 min, followed by a centrifugation step at 20,000×g for 30 min, to eliminate cell debris and apoptotic bodies, respectively. The resulting supernatant was concentrated to a volume of 500 µL using 10 kDa Amicon® ultra centrifugal filter units (Merck Millipore Ltd., Tullagreen, Carrigtwohill, Ireland). EVs were then isolated using size exclusion chromatography (SEC) (qEVoriginal/35 nm, Izon Science Limited, Christchurch, New Zealand). Before EV isolation, the column was pre-washed with 17 mL of filtered Dulbecco’s phosphate-buffered saline (PBS, Sigma Life Science, Dorset, UK) using 0.2 µm Minisart™ syringe (Thermo Fisher Scientific, Loughborough, UK). Subsequently, 500 µL of the concentrated conditioned media sample was introduced through the top of the column. Following sample passage through the column's top filter, PBS was immediately added to the top, and EV fractions 7 to 10, each of 500 µL, were collected as per manufacturer's protocol. EV fractions were then pooled for subsequent characterisation using a ZetaView® nanoparticle tracking analyser (NTA, PMX 120, Particle Metrix GmbH, Inning am Ammersee, Germany), dissociation-enhanced lanthanide fluorescence immunoassay assays (DELFIA; Wallac Oy, Turku, Finland), and scanning electron microscopy (SEM) (Thermo FEI Quanta 200F SEM, Thermo Fisher Scientific, Waltham, MA, USA). GC-IMS Analysis A GC coupled to a FlavourSpec® ion mobility spectrometer (G.A.S. Dortmund, Germany) was used for GC-IMS sample analysis. Prior to analysis, the samples were heated for 45 min at 40°C or 60°C, respectively. An HT2000H autosampler (HTA SRL, Brescia, Italy) was used to automatically collect 80 µL of sample headspace with a 2.5 mL syringe held at 80°C, and inject it into the GC port kept at 80°C. The VOCs present in each EV sample headspace were pre-separated by a GC MXT-5 column of 30 m length × 0.53 mm internal diameter × 1 µm thickness, with 5%/95% phenyl/methylpolysiloxane stationary phase (Restek Corporation, Bellefonte, Pennsylvania, USA) maintained at a constant temperature of 45°C. Nitrogen (purity > 99.9995%), produced by a nitrogen generator (SIRIO 1500, LNI Swissgas, Milano, Italy), was used as carrier gas, with the flow rate set to 5 mL/min for the first 5 min, which was then increased to 50 mL/min in 5 min, and maintained at 50 mL/min for other 5 min. The eluted compounds passed at a constant N2 flow rate of 150 mL/min through a 5.3 cm drift tube operated at 2.7 kV, where they were sorted by their m/z ratio. The IMS analysis was conducted in the positive ion mode.