Characterization of a Cell-Culturing System for the Study of Contact-Independent Extracellular Vesicle Communication.

Appropriate and well-documented in vitro cell-culturing systems are necessary to study the activity and biological function of extracellular vesicles (EVs). The aim of this study was to describe an experimental system, in which dynamic, vesicle-based cell communication can be investigated. A commercially available cell-culturing system was applied to study contact-independent cell communication, which separated two cell populations using a membrane with a pore size of 0.4 µm. The EV exchange characteristics between the two compartments in the culture set-up was preliminarily investigated in a cell-free set-up, and analysed using the Extracellular Vesicle (EV) Array and Nanoparticle Tracking Analysis. The application of the cell-culturing set-up was demonstrated using co-cultures of human primary cells. The effects of the relative placement of the two cell populations on the phenotype of EVs found in the cell supernatant were investigated. The results indicate that this placement can be important for the biological hypothesis that is being investigated. These observations are relevant for short (<24h) as well as long (several days) studies of vesicle-based cell communication. Moreover, the introduced cell-culturing set-up and analytical strategy can be used to study contact-independent vesicle communication in a reproducible manner.

Phenotyping of Leukocytes and Leukocyte-Derived Extracellular Vesicles.

Extracellular vesicles (EVs) have a demonstrated involvement in modulating the immune system. It has been proposed that EVs could be used as biomarkers for detection of inflammatory and immunological disorders. Consequently, it is of great interest to investigate EVs in more detail with focus on immunological markers. In this study, five major leukocyte subpopulations and the corresponding leukocyte-derived EVs were phenotyped with focus on selected immunological lineage-specific markers and selected vesicle-related markers. The leukocyte-derived EVs displayed phenotypic differences in the 34 markers investigated. The majority of the lineage-specific markers used for identification of the parent cell types could not be detected on EVs released from monocultures of the associated cell types. In contrast, the vesicular presentation of CD9, CD63, and CD81 correlated to the cell surface expression of these markers, however, with few exceptions. Furthermore, the cellular expression of CD9, CD63, and CD81 varied between leukocytes present in whole blood and cultured leukocytes. In summary, these data demonstrate that the cellular and vesicular presentation of selected lineage-specific and vesicle-related markers may differ, supporting the accumulating observations that sorting of molecular cargo into EVs is tightly controlled.

Antibody-Based Assays for Phenotyping of Extracellular Vesicles.

Extracellular vesicles (EVs) are a heterogeneous population of membrane-enclosed vesicles. EVs are recognized as important players in cell-to-cell communication and are described to be involved in numerous biological and pathological processes. The fact that EVs are involved in the development and progression of several diseases has formed the basis for the use of EV analysis in a clinical setting. As the interest in EVs has increased immensely, multiple techniques have been developed aiming at characterizing these vesicles. These techniques characterize different features of EVs, like the size distribution, enumeration, protein composition, and the intravesicular cargo (e.g., RNA). This review focuses on techniques that exploit the specificity and sensitivity associated with antibody-based assays to characterize the protein phenotype of EVs. The protein phenotype of EVs can provide information on the functionality of the vesicles and may be used for identification of disease-related biomarkers. Thus, protein profiling of EVs holds great diagnostic and prognostic potential.