Figure 4.

Three method-combination for concentrating EVs derived from cell culture medium. Cells are grown to confluence in 4 × 175 cm² flasks to produce conditioned medium. Conditioned medium is then processed through the differential ultracentrifugation (UC) protocol in order to obtain an initial UC sample (step 1). The initial UC sample is then processed through a size exclusion chromatography column (SEC - Izon qEV single column) in the aim to separate putative EVs from protein contaminants (step 2). Following measurement of the particle and protein concentration, SEC fractions of interest are then pooled together and concentrated using Amicon ultra 0.5 devices (step 3). Final validation experiments confirm the sEVs concentration and the decreased protein contamination of the sample.