The stability of extracellular vesicles (EVs) both before injection and after injection is a critical parameter to investigate. Once produced, extracellular vesicles have to be conserved correctly to ensure that they keep their efficiency for future use. Interestingly, the most used method is the freezing method at -80°C or -20°C that probably leads to the loss of cargo protein by destruction of the membrane by ice crystals (unpublished results). Anyway, most publications were made with frozen extracellular vesicles, meaning that the loss of the cargo is maybe not the critical parameter to look at. In the few published papers about extracellular vesicle conservation, a loss of total protein, of number of EVs, of specific proteins, or a change in morphology was described [78],  without complete reproducibility between papers [79, 80]. No data on the vesicle integrity and loss of cargo was reported. We consider highly unexpected that extracellular vesicles or proteins would disappear following de-freezing, and rather consider that it was an aggregation effect for the extracellular vesicles (2 EVs packed considered as 1 in nanoparticle tracking analysis, or a small EV that explodes in 2 even smaller ones, not detected anymore), and an artifact for the total protein loss of content that cannot disappear (but not for specific protein that can be degraded by proteases for example). One paper reported an interesting potency assay that is in our opinion the critical parameter to investigate [81]. They observed a loss of antibacterial effect once neutrophil derived extracellular vesicls were frozen, probably due to a loss of function of some proteins among the complex mixture of effector proteins in these extracellular vesicles. Knowing the large variability of proteins’ comportment in response to freezing, we predict that most extracellular vesicles, whose effect are mediated by a complex mixture of effectors, would lose a part of their efficiency once frozen (by the denaturation and loss of protein tertiary structure). Whether this loss of efficiency can be limited, and whether it is a real threat is a major question to be solved. New conservation mechanisms have also to be investigated like freeze drying, as well as the effect of conservatives. Freeze drying is on its own a challenge because it can lead to the denaturation of proteins, each of them behaving differently after drying (cryoprotectants like trehalose can be used to limit this effect [82], and lead to a partial loss of function. On another part if the solution dried contains salts, it can also lead to the production of crystals that can destabilize membranes, therefore it can be interesting to resuspend Exosomes in ammonium bicarbonate buffer during the freeze drying process, a solute that can evaporate with water, avoiding the formation of crystals. 

In the end, these processes would allow the development of efficient “off the shelf” extracellular vesicles, ready to be reconstituted/de-frozen and injected in an emergency room. 

[79] L. Issman, B. Brenner, Y. Talmon, A. Aharon, Cryogenic transmission electron microscopy nanostructural study of shed microparticles, PLoS One, 8 (2013) e83680.

[80] J.L. Welch, M.N. Madison, J.B. Margolick, S. Galvin, P. Gupta, O. Martínez-Maza, C. Dash, C.M. Okeoma, Effect of prolonged freezing of semen on exosome recovery and biologic activity, Scientific Reports, 7 (2017) 45034.

[81] Á.M. Lőrincz, C.I. Timár, K.A. Marosvári, D.S. Veres, L. Otrokocsi, Á. Kittel, E. Ligeti, Effect of storage on physical and functional properties of extracellular vesicles derived from neutrophilic granulocytes, Journal of Extracellular Vesicles, 3 (2014) 25465.

[82] S. Bosch, L. de Beaurepaire, M. Allard, M. Mosser, C. Heichette, D. Chrétien, D. Jegou, J.-M. Bach, Trehalose prevents aggregation of exosomes and cryodamage, Scientific Reports, 6 (2016) 36162.

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