Isolation of CD9+ extracellular vesicles from plasma using differential centrifugation and characterization by flow cytometry

Introduction: Extracellular vesicles (EVs) are lipid bilayer vesicles (40-1000nm in diameter) released by all cell types and mediate intercellular communication. Numerous methods for EVs isolation and characterization are available, but their utilization has been limited, partly due to volume constraints and poor reproducibility. Therefore, the aim of this study was to implement a differential centrifugation method for isolating EVs and subsequently identifying surface markers on these EVs through flow cytometry.  

Methods: Blood samples were obtained from four inflamed patients from University Hospital HU/UFSC, using techniques to minimize the EVs formation, such as discarding the first EDTA tube, collecting without a tourniquet and not inverting the tubes. EVs were isolated from the Platelet-Poor Plasma (PPP) by the differential centrifugation technique using a bench centrifuge as described by Laurenzana et al., 2021 with modifications. For characterization of EVs, Dynamic Light Scattering (DLS), Transmission Electron Microscopy (TEM) and conventional Flow Cytometry (FC) were used. EVs were diluted in 0,22 μm PBS for the DLS assay (1:20 V/V) for measure the particle size and polydispersity (PDI). TEM analysis was performed to analyze the EVs presence and their characteristics. BD FACS Canto II calibration was performed and nanopolystyrene size standard kit was used to set up voltages for physical parameters Forward Scatter (FSC) and Side Scatter (SSC). Threshold was set on SSC and samples were diluted in serial dilutions and incubated with EVs markers such as anti-CD9, anti-CD63, anti-CD81 and Annexin V. This study was approved by the local ethics committee (CEPSH/UFSC nº CAAE 75361223.6.0000.0121/2023)

Results: The differential centrifugation allowed the isolation of mainly large EVs (>200 nm). The size of the EVs were confirmed by DLS and TEM assays. In the FC assay, the isolated EVs exhibited the presence of the CD9 marker. This finding was confirmed by the introduction of Triton X-100 (negative control), that cause disappearance of CD9+ events. However, CD81, CD63, and Annexin-V displayed autofluorescence, which shows their inadequacy on the accurate identification of EVs.

Conclusions: These results suggest that the differential centrifugation method can be used for the EVs isolation in laboratories that only have a bench centrifuge. Furthermore, it demonstrates that it is possible to identify and characterize EVs by the FACS Canto II flow cytometer. This allows the study of EVs for various research purposes, such as the evaluation of prognostic biomarkers in neoplasms.

Financial Support: This research was supported by the Brazilian National Council for Scientific and Technological Development (CNPq), Notice CNPq/MCT N° 10/2023 – Universal