The endo-lysosomal system of bEnd.3 and hCMEC/D3 brain endothelial cells

Abstract

Background Brain endothelial cell-based in vitro models are among the most versatile tools in blood–brain barrier research for testing drug penetration to the central nervous system. Transcytosis of large pharmaceuticals across the brain capillary endothelium involves the complex endo-lysosomal system. This system consists of several types of vesicle, such as early, late and recycling endosomes, retromer-positive structures, and lysosomes. Since the endo-lysosomal system in endothelial cell lines of in vitro blood–brain barrier models has not been investigated in detail, our aim was to characterize this system in different models. Methods For the investigation, we have chosen two widely-used models for in vitro drug transport studies: the bEnd.3 mouse and the hCMEC/D3 human brain endothelial cell line. We compared the structures and attributes of their endo-lysosomal system to that of primary porcine brain endothelial cells. Results We detected significant differences in the vesicular network regarding number, morphology, subcellular distribution and lysosomal activity. The retromer-positive vesicles of the primary cells were distinct in many ways from those of the cell lines. However, the cell lines showed higher lysosomal degradation activity than the primary cells. Additionally, the hCMEC/D3 possessed a strikingly unique ratio of recycling endosomes to late endosomes. Conclusions Taken together our data identify differences in the trafficking network of brain endothelial cells, essentially mapping the endo-lysosomal system of in vitro blood–brain barrier models. This knowledge is valuable for planning the optimal route across the blood–brain barrier and advancing drug delivery to the brain. Electronic supplementary material The online version of this article (10.1186/s12987-019-0134-9) contains supplementary material, which is available to authorized users.

Combination of Alanine and Glutathione as Targeting Ligands of Nanoparticles Enhances Cargo Delivery into the Cells of the Neurovascular Unit

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Nanoparticles Based on Quaternary Ammonium Chitosan-methyl-β-cyclodextrin Conjugate for the Neuropeptide Dalargin Delivery to the Central Nervous System: An In Vitro Study

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SorCS2 deletion leads to altered neuronal lysosome activity

“…The total number of vesicles was normalized to the number of nuclei before making comparison among the adjacent groups. Number and mean fluorescent intensity of vesicles from 100 to 500 cells for each group were analyzed (Toth, Nielsen et al 2019).…”

“…Cells were washed twice with D-PBS and kept in FluoroBrite DMEM media (Gibco, A1896701) supplemented with 1% GlutaMAX for imaging. Method described before (Toth, Nielsen et al 2019).…”