Size exclusion columns

Isolation of sEVs from either patient serum/plasma or cell culture conditioned media was performed by size exclusion chromatography or ultrafiltration, respectively, as previously described [31 (link)]. For patient serum/plasma, sEV samples were overlaid on size exclusion columns (Izon Science, Ltd. Christchurch, New Zealand) and eluted with phosphate buffered saline, with sEV-positive fractions collected. The fractions were concentrated in Amicon^® Ultra-4 10 kDa centrifugal filter units (Merck Millipore, MA, USA) by centrifugation at 4000× g at 4 °C, using an Allegra^® X-15R centrifuge (Beckman Coulter, CA, USA). For ultrafiltration, cell culture conditioned media was concentrated with a Centricon Plus-70 Centrifugal Filter (Ultracel-PL Membrane, 100 kDa) device (Merck Millipore, MA, USA), by centrifugation at 3500× g at 4 °C, using an Allegra^® X-15R centrifuge (Beckman Coulter, CA, USA). Recovery of sEVs was performed with a reverse spin at 1000× g for 2 min. The final sEV samples were collected and stored at −80 °C conditions.

Conditioned culture media (CCM) containing EVs were first centrifuged at 800 × g for 10 min to remove cells. Following this, CCM was filtered using a 0.22 µm membrane to remove remaining cell debris and large particles. Subsequently, CCM was concentrated to 20 mL and buffer exchanged into PBS with tangential flow filtration using a Minimate capsule with a 300 kDa membrane. The remaining 20 mL was concentrated to 500 µL using a Centricon Plus‐70 Centrifugal Filter with a 100 kDa cutoff, before being purified using size‐exclusion chromatography as previously described.^[40]Plasma was thawed rapidly and clarified on debris and large vesicles through centrifugation at 1500 × g and 10 000 × g for 10 and 20 min, respectively. A total of 500 µL of clarified plasma was overlaid on size exclusion columns (Izon) followed by elution with PBS. High EV‐containing fractions were collected as previously described,^[40] and concentrated to ≈100 µL using Amicon Ultra‐4 10 kDa nominal molecular weight centrifugal filter units.