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

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-MS Analysis The EV samples were analysed using a Shimadzu GCMS-QP2020 NX equipment (Shimadzu Corporation, Kyoto, Japan). 2 mL of sample was placed into a 20 mL sealed glass vial, which was introduced in an HT2800T headspace autosampler (HTA SRL, Brescia, Italy) for incubation at 40 °C or 60 °C for 1 h. The headspace was then exposed to a divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) solid phase microextraction (SPME) fibre for 1 h, and then desorbed for 3 min into the GC-MS injector hold at 250 °C. Inside the gas chromatograph, the VOCs were separated onto an SH-I-5MS capillary column, with 5% diphenyl / 95% dimethyl polysiloxane stationary phase. The temperature ramp employed was: hold at 50 °C for 3 min; ramp of 6 °C/min until reaching 200 °C; hold at 200 °C for 2 min; ramp of 10 °C/min until reaching 280 °C; hold at 280 °C for 3 min. The mass spectrometer used was a single quadrupole analyser operating in the electron ionisation mode, with an ionisation energy of 70 eV. The temperatures of the transfer line and ion source were kept constant at 250 °C. Fibre cleaning was performed at 270 °C for 15 min before each measurement. The data were acquired in the scan mode, for mass ranges between 30 and 450 m/z. Putative compounds’ identification was carried out by comparing the acquired mass spectra with the NIST20 mass spectral library of the National Institute of Standards and Technology (NIST). PBS solution was analysed as background.