Fig. 3

Receptor-mediated transcytosis of a human Brain Shuttle antibody in blood–brain barrier organoids. a Representative confocal images of blood–brain barrier organoids after incubation with different antibody constructs for 4 h. The upper images show an overlay of brain endothelial cells labelled with an anti-P-gp antibody (magenta), antibody constructs labelled with anti-human IgG Fab fragments (yellow) and nuclei labelled with DAPI (cyan). The intensity of the lower images was scaled to visualize the background intensity in the antibody channel (grey). Scale bars, 100 μm. b Quantification of antibody construct transcytosis into blood–brain barrier organoids. Graph shows boxplots with interquartile ranges and median. Lines show the 5th and 95th percentiles. Differences between non-treated organoids and non-targeting IgG or mouse brain shuttle were not statistically significant (p > 0.6) whereas differences between human Brain Shuttle and non-treated control were statistically significant (**p = 0.002) as evaluated by one-way ANOVA followed by Dunnett’s test for multiple comparisons of 60 organoids per condition in n = 3 independent experiments. c Representative orthogonal cross-sections assembled from confocal planes spanning the whole volume of organoids incubated with human Brain Shuttle for 15, 30 or 120 min. The images on the left show the maximum projection of 10 orthogonal planes located at least 25 μm below the organoid surface. Brain endothelial cells are labelled with an anti-P-gp antibody (magenta), antibody constructs are labelled with anti-human IgG Fab fragments (yellow). Scale bars, 50 μm. The graphs on the right show the mean line profile intensity of human Brain Shuttle (yellow) or P-gp (magenta) fluorescence within organoids. Distance in μm is measured from left to right across the ROI shown within the dotted box. d Time course of human Brain Shuttle transcytosis into the core of blood–brain barrier organoids reconstructed from organoid populations fixed with PFA at different time points. e Dose–response of human Brain Shuttle transcytosis into the core of blood–brain barrier organoids. In d and e points represent individual organoids. The solid lines show the best nonlinear (d) or linear (e) fit of the experimental data. The dotted lines and colored area show the 95% confidence interval of the curve estimation