Background

Mesenchymal stem cells (MSCs) have a protective effect on the progression of different diseases contributing to the immunomodulation and the inflammatory state (Bartholomew et al., 2002; Togel et al., 2005; Azmi et al., 2013; Ebrahimi et al., 2013; Ben-Ami et al., 2014). Their protective action is not only due to their transdifferentiation, but also their paracrine mechanisms such as release of extracellular vesicles (EVs) showing an immunomodulatory, anti-inflammatory, anti-apoptotic and proangiogenic function (Bruno et al., 2009; Camussi et al., 2010a; Grange et al., 2020). Moreover, the application of EVs from MSCs in clinical practice has the advantage of being a safe therapy without the disadvantages related to other MSCs therapies, including the possibility to be rejected, gene stability, poor long-term differentiation and probability of viral transfer (Kunter et al., 2007; Wang et al., 2012).

In the last years, there is an increasing interest in the application of EVs in the transplant area, as an immunomodulatory and anti-inflammatory therapy on transplant recipients, and their addition on perfusion solution to pre-condition the graft before transplantation (Koch et al., 2015; Gregorini et al., 2017; Stone et al., 2017; Rigo et al., 2018; Ramírez-Bajo et al., 2020b). In particular, EVs therapy in the context of a hypothermic or normothermic perfusion machine could increase the long-term survival and increase the number of available organs. This improvement could be related to a decrease of inflammation, the preservation of energy metabolism and microvascular permeability avoiding the formation of edema. These properties show the great therapeutic potential of EVs in the transplantation process.

Cell-to-cell communication by EVs is a mechanism that has been preserved throughout the evolution in both prokaryotic and eukaryotic cells (Ratajczak et al., 2006). Since its discovery 30 years ago, it has been demonstrated that EVs are produced by an enormous variety of cell types such as blood cells, dendritic, endothelial and epithelial cells, nervous system cells, adult and embryonic stem cells and even tumor cells (Harding et al., 1984). EVs are a heterogeneous group of membranous vesicles that differ on their biogenesis and release pathways such as exosomes, microvesicles, microparticles, ectosomes, oncosomes and apoptotic bodies (Thery et al., 2002 and 2018). EVs act as paracrine/endocrine effectors that transport bioactive molecules among the cells (cytosolic proteins, lipids, mRNA, miRNA, DNA, mitochondrial DNA and genomic DNA) of the surrounding microenvironment, or being carried remotely in biological fluids (Fevrier and Raposo, 2004; Camussi et al., 2010b; Mittelbrunn et al., 2011). These regulatory biological signals can regulate various physiological processes, as well as the development and progression of disorders (Valadi et al., 2007; Guescini et al., 2010; Balaj et al., 2011).

A large number of studies are focused on EV’s field and their isolation from different biofluids, developing different methods of isolation such as ultracentrifugation, density gradient, size exclusion chromatography, filtration, microfluidics, precipitation kits and magnetic beads technologies. The choice of isolation methods should depend on the type of starting material, volume, available equipment, scientific inquiry and subsequent analysis or use (Momen-
Heravi et al., 2013). There are described different protocols associated with difference in purity and time of isolation. Moreover, EVs composition is dependent on the isolation protocol used, obtaining different subpopulations with different soluble proteins and nucleic acids. For these reasons, it is important to follow a series of criteria based on current best practice that represents the minimal experimental requirements for definition of EVs that allow the interpretation and their replication by other researchers (Lotvall et al., 2014).

The most commonly used protocol for isolation is ultracentrifugation which has advantages such as the applicability for EVs isolation from large volumes as urine or cell culture conditioned media, the use of common media and buffers, easy to handle, and absence of impact on EVs except gravitational force (Konoshenko et al., 2018). This method can be used to obtain large quantity of EVs with a good reproducibility for developing studies about their biological actions. However, it needs expensive equipment and the isolated EVs could present co-isolating contaminants such as soluble proteins and nucleic acids. In our previous publications, we showed a comparison of the therapeutic effect of MSC and their EVs in an in vivo model of chronic kidney allograft rejection (Ramírez-Bajo et al., 2020a) and chronic cyclosporine nephrotoxicity (Ramírez-Bajo et al., 2020b). The ultracentrifugation protocol allows to produce a great quantity of EVs from high volumes of MSC-conditioned medium required for a periodic administration of therapies to different animal groups. We observed a good reproducibility between EVs batches by this methodology.