Abstract
The research field on extracellular vesicles (EV) has rapidly expanded in recent years due to the therapeutic potential of EV. Adipose tissue human mesenchymal stem cells (ASC) may be a suitable source for therapeutic EV. A major limitation in the field is the lack of standardization of the challenging techniques to isolate and characterize EV. The aim of our study was to incorporate new controls for the detection and quantification of EV derived from ASC and to analyze the applicability and limitations of the available techniques. ASC were cultured in medium supplemented with 5% of vesicles-free fetal bovine serum. The EV were isolated from conditioned medium by differential centrifugation with size filtration (0.2 μm). As a control, non-conditioned culture medium was used (control medium). To detect EV, electron microscopy, conventional flow cytometry, and western blot were used. The quantification of the EV was by total protein quantification, ExoELISA immunoassay, and Nanosight. Cytokines and growth factors in the EV samples were measured by multiplex bead array kit. The EV were detected by electron microscope. Total protein measurement was not useful to quantify EV as the control medium showed similar protein contents as the EV samples. The ExoELISA kits had technical troubles and it was not possible to quantify the concentration of exosomes in the samples. The use of Nanosight enabled quantification and size determination of the EV. It is, however, not possible to distinguish protein aggregates from EV with this method. The technologies for quantification and characterization of the EV need to be improved. In addition, we detected protein contaminants in the EV samples, which make it difficult to determine the real effect of EV in experimental models. It will be crucial in the future to optimize design novel methods for purification and characterization of EV.
Highlights
Extracellular vesicles (EV) are lipid bilayer particles coming either from the inside of a cell or formed directly from its cell membrane, and excreted to the extracellular medium
A major limitation in the field is the lack of standardization of the challenging techniques to isolate and characterize EV.The aim of our study was to incorporate new controls for the detection and quantification of EV derived from Adipose tissue human mesenchymal stem cells (ASC) and to analyze the applicability and limitations of the available techniques
Protein contents of EV were released by protein extraction protocol, isolated EV, or control medium were re-suspended in 200 μl of RIPA buffer (Sigma-Aldrich) with protease inhibitors cocktail (Sigma-Aldrich), and sonicated for 5 min, three times, vortexing in between
Summary
Extracellular vesicles (EV) are lipid bilayer particles coming either from the inside of a cell or formed directly from its cell membrane, and excreted to the extracellular medium. EV participate in cell-to-cell communication by transfer of proteins, bioactive lipids, and nucleic acids [1]. The term EV includes exosomes (30–100 nm), microvesicles (100–1000 nm), and apoptotic bodies (50–5,000 nm) [2]. Apoptotic bodies are shed from dying cells, while microvesicles are shed from the plasma membrane of viable cells. Exosomes are of endocytic origin and are released when multivesicular bodies fuse with the plasma membrane and release their intraluminal vesicles as exosomes to the extracellular milieu [3]. Exosomes have been defined based on size, density (1.12–1.19 g/ml), and expression of specific biomarkers (e.g., tetraspanins, annexin, and heat shock proteins) [4]. Due to the heterogeneity of the various types of vesicles, it has been suggested to call them collectively EV
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