BACKGROUND: Exosomes are emerging targets for biomedical research. However, suitable
methods for the isolation of blood plasma-derived exosomes without impurities have
not yet been described. AIM: Therefore, we investigated the efficiency and purity
of exosomes isolated with potentially suitable methods; differential ultracentrifugation
(UC) and size exclusion chromatography (SEC). METHODS AND RESULTS: Exosomes were isolated
from rat and human blood plasma by various UC and SEC conditions. Efficiency was investigated
at serial UC of the supernatant, while in case of SEC by comparing the content of
exosomal markers of various fractions. Purity was assessed based on the presence of
albumin. We found that the diameter of the majority of isolated particles fell into
the size range of exosomes, however, albumin was also present in the preparations,
when 1h UC at 4 degrees C was applied. Furthermore, with this method only a minor
fraction of total exosomes could be isolated from blood as deduced from the constant
amount of exosomal markers CD63 and TSG101 detected after serial UC of rat blood plasma
samples. By using UC for longer time or with shorter sedimentation distance at 4 degrees
C, or UC performed at 37 degrees C, exosomal yield increased, but albumin impurity
was still observed in the isolates, as assessed by transmission electron microscopy,
dynamic light scattering and immunoblotting against CD63, TSG101 and albumin. Efficiency
and purity were not different in case of using further diluted samples. By using SEC
with different columns, we have found that although a minor fraction of exosomes can
be isolated without significant albumin content on Sepharose CL-4B or Sephacryl S-400
columns, but not on Sepharose 2B columns, the majority of exosomes co-eluted with
albumin. CONCLUSION: Here we show that it is feasible to isolate exosomes from blood
plasma by SEC without significant albumin contamination albeit with low vesicle yield.
