Innovative Visualization and Quantification of Extracellular Vesicles Interaction with and Incorporation in Target Cells in 3D Microenvironments

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.

In Vitro Study of Extracellular Vesicles Migration in Cartilage-Derived Osteoarthritis Samples Using Real-Time Quantitative Multimodal Nonlinear Optics Imaging

“…[ 44 ]. To improve the knowledge in term of EVs incorporation in 3D structures, we recently proposed a microfluidic device where OA chondrocytes were cultured in 3D conditions allowing a clear detection of their tridimensional morphology in a fibrin-based matrix, and compared this system with 2D cultures [ 45 ]. In this model, ASC-EVs injected in the 3D device quickly and homogeneously permeated the intercellular matrix, also far from the injection site, and they were internalized into the chondrocytes.…”

Experimental Therapeutics for the Treatment of Osteoarthritis

“…EVs mediate paracrinic cell-cell communication, being produced by cell membrane budding of donor cells and are internalized by recipient cells including joint-derived cells such as chondrocytes and SF. 54 , 59 , 192 An uptake of ASC-derived EVs by SF and chondrocytes could be observed in in vitro co-culture models of SF and chondrocytes from the same OA patient which differed under 2D and 3D conditions. Interestingly, SFs showed a faster uptake of EVs than chondrocytes.…”

“… 57 In other studies either synovium-derived MSCs and ATDC5 chondrocytes 58 or human SF and articular chondrocytes were co-cultured. 59 The latter study was undertaken to show the exchange of stem cell-derived extracellular vesicles (EVs) under 2D and 3D conditions in regard to OA. 59 One should critically think about the in vitro models in regard to the fact that inflammation is refined to the presence of either TNFα or IL-1β.…”

“… 59 The latter study was undertaken to show the exchange of stem cell-derived extracellular vesicles (EVs) under 2D and 3D conditions in regard to OA. 59 One should critically think about the in vitro models in regard to the fact that inflammation is refined to the presence of either TNFα or IL-1β. Meanwhile it is known that other cytokines and mediators among them IL-6 or IL-18 might contribute to OA pathogenesis.…”

“… 60 Most of the in vitro studies are performed in 2D culture, but there exist differences in cell behavior of 2D and 3D cultured cells. 59 More sophisticated in vitro models are required, particularly more complex co-cultures including several cell types or tissues, eg, addressing also menisci, the osteochondral unit, synovium and the IFP of the osteoarthritic joint to reflect interactions. 41 , 61 …”