Characterization of an iPSC-based barrier model for blood-brain barrier investigations using the SBAD0201 stem cell line.

BACKGROUND: Blood-brain barrier (BBB) models based on primary murine, bovine, and porcine brain capillary endothelial cell cultures have long been regarded as robust models with appropriate properties to examine the functional transport of small molecules. However, species differences sometimes complicate translating results from these models to human settings. During the last decade, brain capillary endothelial-like cells (BCECs) have been generated from stem cell sources to model the human BBB in vitro. The aim of the present study was to establish and characterize a human BBB model using human induced pluripotent stem cell (hiPSC)-derived BCECs from the hIPSC line SBAD0201. METHODS: The model was evaluated using transcriptomics, proteomics, immunocytochemistry, transendothelial electrical resistance (TEER) measurements, and, finally, transport assays to assess the functionality of selected transporters and receptor (GLUT-1, LAT-1, P-gp and LRP-1). RESULTS: The resulting BBB model displayed an average TEER of 5474 ± 167 Ω·cm2 and cell monolayer formation with claudin-5, ZO-1, and occludin expression in the tight junction zones. The cell monolayers expressed the typical BBB markers VE-cadherin, VWF, and PECAM-1. Transcriptomics and quantitative targeted absolute proteomics analyses revealed that solute carrier (SLC) transporters were found in high abundance, while the expression of efflux transporters was relatively low. Transport assays using GLUT-1, LAT-1, and LRP-1 substrates and inhibitors confirmed the functional activities of these transporters and receptors in the model. A transport assay suggested that P-gp was not functionally expressed in the model, albeit antibody staining revealed that P-gp was localized at the luminal membrane. CONCLUSIONS: In conclusion, the novel SBAD0201-derived BBB model formed tight monolayers and was proven useful for studies investigating GLUT-1, LAT-1, and LRP-1 mediated transport across the BBB. However, the model did not express functional P-gp and thus is not suitable for the performance of drug efflux P-gp reletated studies.

Re-evaluation of the hCMEC/D3 based in vitro BBB model for ABC transporter studies.

The blood-brain barrier (BBB) represents one of the biggest hurdles for CNS related drug delivery, preventing permeation of most molecules, and therefore poses a major challenge for researchers in finding effective treatments for CNS diseases. The low permeability of molecules through the BBB is linked on one hand to the extreme tightness by tight junction (TJ) formation limiting the paracellular transport, and on the other hand to the presence of ATP-driven efflux pumps which actively transport unwanted compounds out of the brain. In this study we evaluated the applicability of the immortalized human cell line hCMEC/D3 for ABC transporter studies, focusing on the most expressed ABC transporters at the human BBB: P-glycoprotein (PGP, ABCB1), multidrug resistance protein 4 (MRP4, ABCC4) and breast cancer resistance protein (BCRP, ABCG2). Therefore, a two-step screening method was applied, consisting of a regular uptake assay (96-well format) and bidirectional transport studies, using a transwell system as in vitro simulation of the human BBB. In conclusion, the hCMEC/D3 based in vitro BBB model is well suited to screen drug candidates for ABC transporter interactions on the basis of a regular uptake assay, but in terms of transcellular permeability studies the cell line is limited by a lack of sufficient junctional tightness.

Physicochemical and biopharmaceutical characterization of novel Matrix-Liposomes.

Matrix-Liposomes (MLs) are a very promising solid oral drug delivery system; however, data on their interaction with biological membranes are not available. Here, we describe the quality of MLs manufactured by dual centrifugation. MLs were prepared with a Z-average range of 139 to 160 nm and a PDI of 0.18 to 0.25. To investigate the effect of MLs on intestinal tissue (with and without mucolytic treatment), we then established an ex vivo rat intestine model. The integrity of the epithelial membranes of rat intestine was not affected by the incubation with MLs without or with pre-mucolytic treatment. Tissue samples were also analysed for changes in P-glycoprotein (P-gp) expression and function. The net secretion of the P-gp substrate Rh123 across the rat duodenum was increased in the presence of MLs. To summarize, MLs do not affect intestinal epithelial integrity, although they impact Rh123 secretion. In future, these novel MLs have to be further evaluated for proficient intestinal drug delivery.