A novel affinity-based method for the isolation of highly purified extracellular vesicles

Extracellular vesicles (EVs) such as exosomes and microvesicles serve as messengers of intercellular network, allowing exchange of cellular components between cells. EVs carry lipids, proteins, and RNAs derived from their producing cells, and have potential as biomarkers specific to cell types and even cellular states. However, conventional methods (such as ultracentrifugation or polymeric precipitation) for isolating EVs have disadvantages regarding purity and feasibility.

Here, Osaka University researchers have developed a novel method for EV purification by using Tim4 protein, which specifically binds the phosphatidylserine displayed on the surface of EVs. Because the binding is Ca2+-dependent, intact EVs can be easily released from Tim4 by adding Ca2+ chelators. Tim4 purification, which the researchers have applied to cell conditioned media and biofluids, is capable of yielding EVs of a higher purity than those obtained using conventional methods. The lower contamination found in Tim4-purified EV preparations allows more EV-specific proteins to be detected by mass spectrometry, enabling better characterization and quantification of different EV populations’ proteomes. Tim4 protein can also be used as a powerful tool for quantification of EVs in both ELISA and flow cytometry formats. Thus, the affinity of Tim4 for EVs will find abundant applications in EV studies.

Evaluation of sEVs and large EVs

(a) sEVs (1 × 10¹⁰ particles) from 10K sup of K562 cells (serum free) isolated by each method were placed on a carbon-coated grid, and allowed to be absorbed onto the grid for 3 min. After being rinsed with distilled water, the sample was stained with a 2% aqueous uranyl acetate solution and examined with a JEOL JEM1011 electron microscope operating at 80 kV. Scale bar, 200 nm. (b) Isolated sEVs were diluted to a suitable concentration with PBS, and the size distribution of sEVs was analyzed by NTA using NanoSight LM10. Red error bars indicate +/−1 standard error of the mean. (c) 10K pellet of K562 cell conditioned medium was resuspended in 4 ml RPMI1640 supplemented with 2 mM CaCl₂, and large EVs were isolated by Tim4-affinity method. The size distribution of the vesicles was analyzed by NTA. (d) Large EVs were isolated by each method from 10K pellet, and equal amount of proteins (100 ng) were subjected to SDS-PAGE, and analyzed by western blot against an EV marker (LAMP2). The flow through of 10K pellet after Tim4-affinity purification was further ultracentrifuged, and the same proportion of the protein amount to the total purified protein amount as Tim4-affinity purification was loaded to detect residual large EVs (Tim4 FT → UC).