Abstract
Extracellular vesicles (EVs) showed therapeutic properties in several applications, many in regenerative medicine. A clear example is in the treatment of osteoarthritis (OA), where adipose-derived mesenchymal stem cells (ASCs)-EVs were able to promote regeneration and reduce inflammation in both synovia and cartilage. A still obscure issue is the effective ability of EVs to be internalized by target cells, rather than simply bound to the extracellular matrix (ECM) or plasma membrane, since the current detection or imaging technologies cannot fully decipher it due to technical limitations. In the present study, human articular chondrocytes (ACHs) and fibroblast-like synoviocytes (FLSs) isolated from the same OA patients were cocultured in 2D as well as in 3D conditions with fluorescently labeled ASC-EVs, and analyzed by flow cytometry or confocal microscopy, respectively. In contrast with conventional 2D, in 3D cultures, confocal microscopy allowed a clear detection of the tridimensional morphology of the cells and thus an accurate discrimination of EV interaction with the external and/or internal cell environment. In both 2D and 3D conditions, FLSs were more efficient in interacting with ASC-EVs and 3D imaging demonstrated a faster uptake process. The removal of the hyaluronic acid component from the ECM of both cell types reduced their interaction with ASC-EVs only in the 2D system, showing that 2D and 3D conditions can yield different outcomes when investigating events where ECM plays a key role. These results indicate that studying EVs binding and uptake both in 2D and 3D guarantees a more precise and complementary characterization of the molecular mechanisms involved in the process. The implementation of this strategy can become a valuable tool not only for basic research, but also for release assays and potency prediction for clinical EV batches.
Highlights
Extracellular vesicles (EVs) are nanoparticles delimited by a lipid bilayer that are naturally released from almost all cell types [1]
adipose-derived mesenchymal stem cells (ASCs)-EVs mostly ranged between 50 nM and 400 nM (Figure 2B), with the majority of particles resulting in smaller than 200 nM
The absence of EVs major protein contamination, that can influence size determination with particle aggregates, was confirmed by protein analysis of EV batches that resulted to have 0.33 × 109 ± 0.12 particles/μg protein (N = 4, mean ± SEM), falling in the 108 to 1010 range claimed for pure preparations [16]
Summary
Extracellular vesicles (EVs) are nanoparticles delimited by a lipid bilayer that are naturally released from almost all cell types [1] Particles differ in both size and origin, either endosomal or plasma membrane-derived [2]. Endosomal EVs are generally smaller (50–150 nM) and referred to as exosomes, whereas plasma-membrane EVs are larger (>100 nM) and defined as microvesicles The difference in their origin suggested a differential fingerprint, for both cargo and functions [2], to date, no molecular signatures have been defined to sharply distinguish these two categories of nanoparticles. EVs act as intercellular messengers to transfer biological signals such as nucleic acids, proteins and lipids [4] Due to this function of cargo transfer, EVs have been recently studied as therapeutics for the delivery of both natural and engineered loads [5]. In osteoarthritis (OA), characterized by both degenerative and inflammatory processes, MSC-EVs were able to promote the regeneration of joint components, such as synovium and cartilage, and to reduce pain [7] through the delivery of their cargo enriched in anti-inflammatory and trophic factors and miRNAs [8]
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