Extracellular vesicles (EVs) have become an attractive field among the scientific community. Yet, a major challenge is to define a consensus method for EVs isolation. Ultracentrifugation has been the most widely used methodology but rapid methods, including Size Exclusion Chromatography (SEC) and/or precipitating agents such as Polyethylene glycol (PEG) or PRotein Organic Solvent PRecipitation (PROSPR) have emerged. To evaluate the impact of these different methods on the resulting EV preparations, plasma EVs were isolated using SEC, PEG and PROSPR and their total protein content, NTA and Cryo-electron microscopy profiles and EV-markers were compared. Also, their effect on recipient cells was tested. Low protein content and Cryo-EM analysis showed that SEC removed most of the overabundant soluble plasma proteins, which were not removed using PEG and partially by PROSPR. Moreover, only SEC allowed the detection of the EV-markers CD9, CD63 and CD81, LGALS3BP and CD5L, suggesting a putative interference of the precipitating agents in the structure/composition of the EVs. Furthermore, PEG and PROSPR-based EV isolation resulted in reduced cell viability in vitro. These results stress that appropriate EV-isolation method should be considered depending on the forthcoming application of the purified EVs.

Undesired immune responses have drastically hampered outcomes after allogeneic organ transplantation and cell therapy, and also lead to inflammatory diseases and autoimmunity. Umbilical cord mesenchymal stem cells (UCMSCs) have powerful regenerative and immunomodulatory potential, and their secreted extracellular vesicles (EVs) are envisaged as a promising natural source of nanoparticles to increase outcomes in organ transplantation and control inflammatory diseases. However, poor EV preparations containing highly-abundant soluble proteins may mask genuine vesicular-associated functions and provide misleading data. Here, we used Size-Exclusion Chromatography (SEC) to successfully isolate EVs from UCMSCs-conditioned medium. These vesicles were defined as positive for CD9, CD63, CD73 and CD90, and their size and morphology characterized by NTA and cryo-EM. Their immunomodulatory potential was determined in polyclonal T cell proliferation assays, analysis of cytokine profiles and in the skewing of monocyte polarization. In sharp contrast to the non-EV containing fractions, to the complete conditioned medium and to ultracentrifuged pellet, SEC-purified EVs from UCMSCs inhibited T cell proliferation, resembling the effect of parental UCMSCs. Moreover, while SEC-EVs did not induce cytokine response, the non-EV fractions, conditioned medium and ultracentrifuged pellet promoted the secretion of pro-inflammatory cytokines by polyclonally stimulated T cells and supported Th17 polarization. In contrast, EVs did not induce monocyte polarization, but the non-EV fraction induced CD163 and CD206 expression and TNF-α production in monocytes. These findings increase the growing evidence confirming that EVs are an active component of MSC's paracrine immunosuppressive function and affirm their potential for therapeutics in nanomedicine. In addition, our results highlight the importance of well-purified and defined preparations of MSC-derived EVs to achieve the immunosuppressive effect.

Mesenchymal stem or stromal cells (MSC) have proven immunomodulatory properties toward B cell activation and induce regulatory B cells (Breg), through a dual mechanism of action that relies both on cell contact and secreted factors. One of them are MSC-derived extracellular vesicles (EVs), membrane nanovesicles that mediate cell communication and typically reflect the phenotype of the cell of origin. MSC-EVs could resemble MSC functions, and are being contemplated as an improved alternative to the MSC-based immunomodulatory therapy. In the present work, we focused on the factors secreted by MSC and aimed to elucidate the putative role of MSC-EVs in the immunomodulation of B cells. EVs and soluble protein-enriched fractions (PF) were isolated from MSC-conditioned medium (CM) using size-exclusion chromatography (SEC) and their capacity to modulate B cell activation, induction of Breg and B cell proliferation was compared to that of the whole MSCs. Co-culture with MSC or unfractionated CM induced naïve and CD24^hiCD38^hi, IL-10 producing (Breg) phenotypes on B cells while not affecting proliferation. MSC-PF had a comparable effect to MSCs, inducing a naïve phenotype, and even though they did not induce the shift toward a CD24^hiCD38^hi population, MSC-PF fostered IL-10 production by B cells. Conversely, MSC-EVs failed to promote naïve B cells and to reduce memory B cells. MSC-EVs induced CD24^hiCD38^hi B cells to a similar extent of that of MSC, but not bona fide Bregs since they did not produce IL-10. Our results show that B cell modulation by MSC is partially mediated by soluble factors other than EVs.

Extracellular vesicles (EVs) large‐scale production is a crucial point for the translation of EVs from discovery to application of EV‐based products. In October 2021, the International Society for Extracellular Vesicles (ISEV), along with support by the FET‐OPEN projects, “The Extracellular Vesicle Foundry” (evFOUNDRY) and “Extracellular vesicles from a natural source for tailor‐made nanomaterials” (VES4US), organized a workshop entitled “massivEVs” to discuss the potential challenges for translation of EV‐based products. This report gives an overview of the topics discussed during “massivEVs”, the most important points raised, and the points of consensus reached after discussion among academia and industry representatives. Overall, the review of the existing EV manufacturing, upscaling challenges and directions for their resolution highlighted in the workshop painted an optimistic future for the expanding EV field.