Fig. 2

Standardized imaging workflow to assess transport into blood–brain barrier organoids. a Scheme and image showing the morphology of a blood-brain barrier organoid mounted on a glass slide and the relative position of imaging planes for experimental acquisition (left). Scale bar, 50 μm. Panels on the right show representative confocal images acquired at the organoid surface and core positions. Scale bar, 100 μm. In all images, brain endothelial cells are labelled with an anti-P-gp antibody (magenta) and are distributed at the organoid surface. Nuclei are labelled with DAPI (cyan). Schematic created with BioRender. b Scheme representing the Z-stack acquisition and analysis process (maximum intensity projection, segmentation and ROI definition) to quantify fluorescent molecules within the organoid core. In all images, nuclei are labelled with DAPI (cyan). Scale bar, 100 μm. c Representative confocal images of blood–brain barrier organoids incubated with different molecular weight Dextrans for 4 h. The upper images show an overlay of Dextran (red) and nuclei labelled with DAPI (cyan). The intensity of the lower images was scaled to visualize the background intensity in the Dextran channel (grey). Scale bars, 100 μm. d Quantification of Dextran fluorescence intensity within blood–brain barrier organoids. Graph shows boxplots with interquartile ranges and median. Lines show the 5th and 95th percentiles. Differences between treatments were not statistically significant (p = 0.51) as evaluated by the non-parametric Friedman test of 50 organoids per condition in n = 4 independent experiments