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Background  In vitro models based on brain capillary endothelial cells (BCECs) are among the most versatile tools in blood–brain barrier research for testing drug penetration into the brain and how this is affected by efflux transporters such as P-glycoprotein (Pgp). However, compared to freshly isolated brain capillaries or primary BCECs, the expression of Pgp in immortalized BCEC lines is markedly lower, which prompted us previously to transduce the widely used human BCEC line hCMEC/D3 with a doxycycline-inducible  MDR1 -EGFP fusion plasmid. The EGFP-labeled Pgp in these cells allows studying the localization and trafficking of the transporter and how these processes are affected by drug exposure. Here we used this strategy for the rat BCEC line RBE4 and performed a face-to-face comparison of RBE4 and hCMEC/D3 wild-type (WT) and  MDR1 -EGFP transduced cells.    Methods   MDR1 -EGFP-transduced variants were derived from WT cells by lentiviral transduction, using an  MDR1 -linker-EGFP vector. Localization, trafficking, and function of Pgp were compared in WT and  MDR1 -EGFP transduced cell lines. Primary cultures of rat BCECs and freshly isolated rat brain capillaries were used for comparison.    Results  All cells exhibited typical BCEC morphology. However, significant differences were observed in the localization of Pgp in that RBE4- MDR1 -EGFP cells expressed Pgp primarily at the plasma membrane, whereas in hCMEC/D3 cells, the Pgp-EGFP fusion protein was visible both at the plasma membrane and in endolysosomal vesicles. Exposure to doxorubicin increased the number of Pgp-EGFP-positive endolysosomes, indicating a lysosomotropic effect. Furthermore, lysosomal trapping of doxorubicin was observed, likely contributing to the protection of the cell nucleus from damage. In cocultures of WT and  MDR1 -EGFP transduced cells, intercellular Pgp-EGFP trafficking was observed in RBE4 cells as previously reported for hCMEC/D3 cells. Compared to WT cells, the  MDR1 -EGFP transduced cells exhibited a significantly higher expression and function of Pgp. However, the junctional tightness of WT and  MDR1 -EGFP transduced RBE4 and hCMEC/D3 cells was markedly lower than that of primary BCECs, excluding the use of the cell lines for studying vectorial drug transport.    Conclusions  The present data indicate that  MDR1 -EGFP transduced RBE4 cells are an interesting tool to study the biogenesis of lysosomes and Pgp-mediated lysosomal drug trapping in response to chemotherapeutic agents and other compounds at the level of the blood–brain barrier.    Supplementary Information  The online version contains supplementary material available at 10.1186/s12987-021-00266-z.

fluids and barriers of the cns

Similarities and differences in the localization, trafficking, and function of P-glycoprotein in MDR1-EGFP-transduced rat versus human brain capillary endothelial cell lines