Ultracentrifugation versus kit exosome isolation: nanoLC–MS and other tools reveal similar performance biomarkers, but also contaminations

Aim: For isolation of exosomes, differential ultracentrifugation and an isolation kit from a major vendor were compared. Materials & methods: ‘Case study’ exosomes isolated from patient-derived cells from glioblastoma multiforme and a breast cancer cell line were analyzed. Results: Transmission electron microscopy, dynamic light scattering, western blotting, and so forth, revealed comparable performance. Potential protein biomarkers for both diseases were also identified in the isolates using nanoLC–MS. Western blotting and nanoLC–MS also revealed negative exosome markers regarding both isolation approaches. Conclusion: The two isolation methods had an overall similar performance, but we hesitate to use the term ‘exosome isolation’ as impurities may be present with both isolation methods. NanoLC–MS can detect disease biomarkers in exosomes and is useful for critical assessment of exosome enrichment procedures.
Lay abstract Exosomes are small vesicles that are released from biological cells. Exosomes are viewed as being tools for intracellular communication, and there is evidence that cancer cells can release exosomes to, for example, enable metastasis. Exosomes may be very useful for diagnostics, as the detection and characterization of exosomes may allow disease detection at early stages. However, exosomes must be isolated from blood or other biomaterials prior to analysis. This can be a challenging task, and we have here taken a critical look at two familiar isolation techniques, using samples from breast cancer cell lines and brain cancer cells (glioblastoma). We find that the two approaches have comparable performance, and contain protein biomarkers of their cells of origin. However, the isolated exosomes can contain contaminations, which may cloud the analysis in clinical settings. Hence, there is room for new approaches for the isolation of exosomes.