Volatolomic Analysis of Extracellular Vesicles Extracted from Cultured Cells (eNose)

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). Electronic Nose Analysis Electronic nose analysis of EV headspace was conducted using the last version of the gas sensor array developed by Tor Vergata University of Rome, Italy. The device consists of an ensemble of ten quartz crystal microbalances (QMBs) placed in a measurement chamber of ~8 cm3 volume. The QMB sensors were coated with molecular films made of porphyrinoids. The gas sensors were complemented by a temperature and relative humidity sensor (SHT31, Sensirion, Switzerland), which allowed monitoring the operating conditions. Vapour samples delivery was controlled by a low noise, high precision, durable miniature diaphragm pump, together with a three-way electron valve incorporated inside the electronic nose. The provision of the electron valve and a flow sensor (Sensor Airflow 200 sccm short 3V, Honeywell, Charlotte, USA) guaranteed a constant flow rate during sample measurement. The electronic nose system was connected and powered via USB from a personal computer (PC). The operation and data acquisition processes were controlled by an in-house developed software running in Matlab.