Impact of Experimental Conditions on Extracellular Vesicles’ Proteome: A Comparative Study

Abstract

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.

Extracellular Vesicles for Therapeutic Nucleic Acid Delivery: Loading Strategies and Challenges

“…Although some external stimuli (e.g., cell starvation, hypoxia, or an increase in Ca 2+ concentration in the culture medium) are used to increase the secretion of EVs [ 105 ], the composition and properties of such EVs can differ significantly from those obtained under normal conditions. This has recently been confirmed for EVs secreted by starving cells, where starvation significantly affects the protein composition of EVs [ 106 ]. The altering of the EV content in response to environmental stresses has also been discussed in a recently published review [ 107 ].…”

Extracellular Vesicles: A New Star for Gene Drug Delivery

“…Therefore, during cell culture, culture medium with exosome-free FBS can be used to generate relatively pure EVs. 83 Similarly, glucose starvation method can also increase the secretion of EVs from cells. For example, the quantity of EVs released by cardiac cells was enhanced by the depletion of glucose in the cell culture medium, suggesting the importance of glucose in regulating the EVs generation process.…”