Exosomes are gaining a prominent role in research due to their intriguing biology and several therapeutic opportunities. However, their accurate purification from body fluids and detailed physicochemical characterization remain open issues. We isolated exosomes from serum of patients with Multiple Myeloma by four of the most popular purification methods and assessed the presence of residual contaminants in the preparations through an ad hoc combination of biochemical and biophysical techniques - including Western Blot, colloidal nanoplasmonics, atomic force microscopy (AFM) and scanning helium ion microscopy (HIM). The preparations obtained by iodixanol and sucrose gradients were highly pure. To the contrary, those achieved with limited processing (serial centrifugation or one step precipitation kit) resulted contaminated by a residual matrix, embedding the exosomes. The contaminated preparations showed lower ability to induce NfkB nuclear translocation in endothelial cells with respect to the pure ones, probably because the matrix prevents the interaction and fusion of the exosomes with the cell membrane. These findings suggest that exosome preparation purity must be carefully assessed since it may interfere with exosome biological activity. Contaminants can be reliably probed only by an integrated characterization approach aimed at both the molecular and the colloidal length scales.

The term secretome should be expanded to also include the great variety of nanoparticles secreted by cells into the extracellular space, which ranges from lipoproteins of a few nanometers to extracellular vesicles of hundreds of nanometers.

Mesenchymal stromal/stem cells (MSCs) are increasingly employed for tissue regeneration, largely mediated through paracrine actions. Currently, extracellular vesicles (EVs) released by MSCs are major mediators of these paracrine effects. We evaluated whether rat-bone-marrow-MSC-derived EVs (rBMSCs-EVs) can ameliorate tendon injury in an in vivo rat model. Pro-collagen1A2 and MMP14 protein are expressed in rBMSC-EVs, and are important factors for extracellular-matrix tendon-remodeling. In addition, we found pro-collagen1A2 in rBMSC-EV surface-membranes by dot blot. In vitro on cells isolated from Achilles tendons, utilized as rBMSC -EVs recipient cells, EVs at both low and high doses induce migration of tenocytes; at higher concentration, they induce proliferation and increase expression of Collagen type I in tenocytes. Pretreatment with trypsin abrogate the effect of EVs on cell proliferation and migration, and the expression of collagen I. When either low- or high-dose rBMSCs-EVs were injected into a rat-Achilles tendon injury-model (immediately after damage), at 30 days, rBMSC-EVs were found to have accelerated the remodeling stage of tendon repair in a dose-dependent manner. At histology and histomorphology evaluation, high doses of rBMSCs-EVs produced better restoration of tendon architecture, with optimal tendon-fiber alignment and lower vascularity. Higher EV-concentrations demonstrated greater expression of collagen type I and lower expression of collagen type III. BMSC-EVs hold promise as a novel cell-free modality for the management of tendon injuries.

This protocol paper describes how to assign a purity grade and to subsequently titrate extracellular vesicle (EV) solutions of a few microliters in volume by microplate COlorimetric NANoplasmonic (CONAN) assay. The CONAN assay consists of a solution of gold nanoparticles (AuNPs) into which the EV preparation is added. The solution turns blue if the EV preparation is pure, whereas it stays red if soluble exogenous single and aggregated proteins (SAPs; often referred to as protein contaminants) are present. The color change is visible by the naked eye or can be quantified by UV-Vis spectroscopy, providing an index of purity (a unique peculiarity to date). The assay specifically targets SAPs, and not the EV-related proteins, with a detection limit <50 ng/μl (an order of magnitude higher resolution than that of the Bradford protein assay). For pure solutions, the assay also allows for determining the EV number, as the color shift is linearly dependent on the AuNP/EV molar ratio. Instead, it automatically reports if the solution bears SAP contaminants, thus avoiding counting artifacts. The CONAN assay proves to be robust and reliable and displays very interesting performances in terms of cost (inexpensive reagents, run by standard microplate readers), working volumes (1–2 μl of sample required), and time (full procedure takes <1 h). The assay is applicable to all classes of natural and artificial lipid microvesicles and nanovesicles.