Correspondence: Winfried Neuhaus (winfried.neuhaus@ait.ac.at)
*equally contributed
Fluids and Barriers of the CNS 2023, 20(Suppl 1): A23
Small extracellular vesicles (sEVs) have a pivotal role in cell-to-cell communication, they are potential biomarkers and omnipresent in different body fluids. However, establishing methods for proper discrimination between sEVs and other particles is challenging [1]. Improvement of methods is highly needed to optimise the steps of sEV workflow for in vitro studies regarding the blood–brain barrier (BBB): from sEV isolation, quality, labelling, characterisation, to permeability and cell-EV interaction studies using e.g., transwell inserts. After sEV isolation (size exclusion chromatography, ultrafiltration) under adequate storage conditions (− 80 °C), discrimination between bona fide sEVs and other particles is essential [2]. Small EVs were fluorescently labelled (cell transfection, lipid-tracer dyes, membrane permeable dyes), simultaneously characterised by Western blotting for sEV markers; light scattering (NTA, flow cytometry) for concentration, size distribution, zeta potential; and sensitive microscopy-based methods for single particle analysis (correlative fluorescence cryo-electron microscopy, EVQuant®) [2]. Two BBB models were used: hCMEC/D3 immortalised cell line [3] and human induced pluripotent stem cell-derived brain capillary endothelial-like cells, hiPSC-BCECs [4]. EV labelling studies have revealed the importance of specific controls for single steps of EV preparation (controls of EV permeation, dye labelling, collection medium). Matrix coating volume and composition, properties of inserts (porosity, pore sizes) and the presence of serum are among the main influencing factors for permeation through both blanks and cell layers. Less than 1% of fluorescent EVs obtained with optimised protocols permeated across hCMEC/D3 and hiPSC-BCEC layers. Method development is indispensable to gain comparable results on EV permeation and EV interaction with cells in transwell BBB models. Controls are key elements since not all particles that scatter are EVs.
Grant Support: This work was founded by the European Union’s Horizon 2020 research and innovation programme (Marie Skłodowska-Curie project No 860303). We further gratefully acknowledge the financial support provided by the Austrian Science Fund FWF (project P 34137-B).