Fig. 3
PD-EEVs cross the human BBB chip. (A-B) Confocal images of the endothelial vessel in the BBB chip showing BMEC-like cells (phalloidin, white) and the presence of EEVs (CellMask orange) in the abluminal side of the vessel (white arrows) (A) and within the endothelial cells (B) 48 h post-treatment. Scale bars: 50 μm (A), 20 μm (B). (C) Quantification of 3 kDa dextran-TMRE apparent permeability (Paap) values in BBB chips treated with vehicle control (i.e. untreated), control EEVs, mild or PD EEVs. (D) Nanoparticle FACS-based quantification of EEVs in conditioned media collected from the vascular or brain compartments. EEVs quantified in the brain compartment have transcytosed from within the luminal side of the endothelial vessel. In (C) and (D), each point represents a control vessel (line ID #38554) untreated (n = 3) or treated with EEVs from control subjects (n = 6 different donors, mean age = 68±5), or patients with mild (n = 4–5 different donors, UPDRS = 29±4, mean age = 59±6) or severe (n = 5 different donors, UPDRS = 80±10, mean age = 67±7) clinical manifestations according to the UDPRS scale. Statistical analysis: Error bars represent mean + SEM. Statistical analysis was performed using Wilcoxon signed rank test with theoretical median (C – untreated; D – Ctrl EEV) set at 1 (*p-value < 0.05; **p-value < 0.01). Abbreviations: Ctrl, control; DAPI, 4′,6-diamidino-2-phenylindole; EEV, extracellular vesicles derived from erythrocytes; PD, Parkinson’s disease