Extracellular vesicle (EV) research is a rapidly developing field, mainly due to the
key role of EVs in intercellular communication and pathophysiological processes. However,
the heterogeneity of EVs challenges their exploration and the establishment of gold-standard
methods. Here, we aimed to reveal the influence of technical changes on EV biology
and the reliability of experimental data. We used B16F1 melanoma cells as a model
and applied nanoparticle tracking analysis, mass spectrometry (LC-MS/MS) and pathway
enrichment analysis to analyze the quantity, size distribution, proteome and function
of their small EVs (sEVs) produced in sEV-depleted fetal bovine serum (FBS)-containing
medium or serum-free medium. Additionally, we investigated the effects of minor technical
variances on the quality of sEV preparations. We found that storage of the isolates
at −80 °C has no adverse effect on LC-MS/MS analysis, and an additional washing step
after differential ultracentrifugation has a minor influence on the sEV proteome.
In contrast, FBS starvation affects the production and proteome of sEVs; moreover,
these vesicles may have a greater impact on protein metabolism, but a smaller impact
on cell adhesion and membrane raft assembly, than the control sEVs. As we demonstrated
that FBS starvation has a strong influence on sEV biology, applying serum-free conditions
might be considered in in vitro sEV studies.
