Ready-made chromatography columns for extracellular vesicle isolation from plasma

Abstract

Proteomic studies of circulating vesicles are hampered by difficulties in purifying vesicles from plasma and serum. Isolations are contaminated with high-abundance blood proteins that may mask genuine vesicular-associated proteins and/or simply provide misleading data. In this brief report, we explored the potential utility of a commercially available size exclusion chromatography column for rapid vesicle purification. We evaluated the performance of the column, with cancer cell line conditioned medium or healthy donor plasma, in terms of removing non-vesicular protein and enriching for vesicles exhibiting exosome characteristics. Serial fractions revealed a peak for typical exosomal proteins (CD9, CD81 etc.) that preceded the peak for highly abundant proteins, including albumin, for either sample type, and harvesting only this peak would represent elimination of >95% of protein from the sample. The columns showed good reproducibility, and streamlining the workflow would allow the exosome-relevant material to be collected in less than 10 minutes. Surprisingly, however, subsequent post-column vesicle concentration steps whilst resulting in some protein loss also lead to low vesicle recoveries, with a net effect of reducing sample purity (assessed by the particle-to-protein ratio). The columns provide a convenient, reproducible and highly effective means of eliminating >95% of non-vesicular protein from biological fluid samples such as plasma.

• exosomes
• plasma
• chromatography
• nanoparticle tracking
• exosome precipitation
• ultracentrifugation

To access the supplementary material to this article, please see Supplementary files under ‘Article Tools’.

To access the supplementary material to this article, please see Supplementary files under ‘Article Tools’.

Acknowledgements

The Cardiff Exosome group is funded by a Programme Grant awarded from Cancer Research Wales (AC), by Prostate Cancer-UK (AC & JPW), by Movember (GAP-1) (AC) and by the MS Society (JLW).

Conflict of interest and funding

MJ and LB are employees of Cell Guidance Systems, Cambridge, UK. The authors (JW, JPW and AC) have not received any funding benefits from CellGS other than the supply of the columns to test.

Notes

To access the supplementary material to this article, please see Supplementary files under ‘Article Tools’.