Exosome Isolation by Ultracentrifugation and Precipitation: A Comparison of Techniques for Downstream Analyses

Exosomes are 50-150 nm diameter extracellular vesicles secreted by all mammalian cell, except mature red blood cells, and contribute to diverse physiological and pathological functions within the body. Many methods have been used to isolate and analyze exosomes, resulting in inconsistencies across experiments and raising questions about how to compare results obtained using different approaches. Questions have also been raised regarding the purity of the various preparations with regard to the sizes and types of vesicles, and to the presence of lipoproteins. Thus, investigators often find it challenging to identify the optimal exosome isolation protocol for their experimental needs. Our laboratories have compared ultracentrifugation and commercial precipitation and column-based exosome isolation kits for exosome preparation. Here, we present our study of exosomes isolated using two of the most commonly-used methods, ultracentrifugation and precipitation, followed by downstream analyses.. We used NanoSight nanoparticle tracking analysis (NTA) and Flow cytometry (Cytek ® ) to determine exosome concentrations and size. Imaging flow cytometry can be utilized to both size and immunophenotype surface markers on exosomes (ImageStream). High-performance liquid chromatography (HPLC) followed by nano-flow liquid chromatography mass spectrometry (LCMS) of the exosome fractions can be used to determine the presence of lipoproteins, with LCMS able to provide a proteomic profile of the exosome preparations. We found that the precipitation method was six times faster, and resulted in ~2.5 fold higher concentration of exosomes per ml compared to ultracentrifugation. Both methods yielded EVs in the size range of exosomes and both preparations included apoproteins.