Porcine Brain Microvessel Endothelial Cells as an in Vitro Model to Predict in Vivo Blood-Brain Barrier Permeability

Abstract

ABSTRACT:The objective of the study was to establish primary cultured porcine brain microvessel endothelial cells (PBMECs) as an in vitro model to predict the blood-brain barrier (BBB) permeability in vivo. The intercellular tight junction formation of PBMECs was examined by electron microscopy and measured by transendothelial electrical resistance (TEER). The mRNA expression of several BBB transporters in PBMECs was determined by reverse transcriptionpolymerase chain reaction analysis. The in vitro permeability of 16 structurally diverse compounds, representing a range of passive diffusion and transporter-mediated mechanisms of brain penetration, was determined in PBMECs. Except for the perfusion flow rate marker diazepam, the BBB permeability of these compounds was determined either in our laboratory or as reported in literature using in situ brain perfusion technique in rats. Results in the present study showed that PBMECs had a high endothelium homogeneity, an mRNA expression of several BBB transporters, and high TEER values. Culturing with rat astrocyte-conditioned medium increased the TEER of PBMECs, but had no effect on the permeability of sucrose, a paracellular diffusion marker. The PB-MEC permeability of lipophilic compounds measured under stirred conditions was greatly increased compared with that measured under unstirred conditions. The PBMEC permeability of the 15 test compounds, determined under the optimized study conditions, correlated with the in situ BBB permeability with an r 2 of 0.60.Removal of the three system L substrates increased the r 2 to 0.89.In conclusion, the present PBMEC model may be used to predict or rank the in vivo BBB permeability of new chemical entities in a drug discovery setting.One major hurdle of successful central nervous system drug delivery is to penetrate the blood-brain barrier (BBB) to reach the therapeutic targets. The BBB is a continuous layer of endothelial cells that are connected to each other through tight junctions. In contrast to the endothelial cells of peripheral blood vessels, the brain microvessel endothelial cells are characterized by unique intercellular tight junctions, the absence of fenestrations, and minimal pinocytic activity. The BBB represents a physiological barrier that efficiently restricts free paracellular passage of most substances from the blood to the brain extracellular environment. Furthermore, the brain microvessel endothelial cells possess a variety of metabolic enzyme systems, which further limit the brain entry of compounds (Pardridge, 1983). Finally, a complex of membrane-bound transport systems, including active efflux transporters, such as P-glycoprotein (Pgp) (Schinkel et al., 1996;Miller et al., 2000) and multidrug resistanceassociated protein (MRP) Zhang et al., 2000), and active uptake transporters, such as the system L amino acid transporter (LAT) (Pardridge, 1983;Smith, 1991), further regulates brain penetration.Pharmaceutical companies have been actively pursuing various methods to accurately predict the brain penetrati...

Setting-up an <em>In Vitro</em> Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport

“…Brain endothelial cells have been extracted from various species to implement in vitro BBB models based on low passage primary cultures from bovine 6,7 , porcine 8,9 . In vitro BBB models are usually used to compare the transport of drugs with: i) known penetration into the CNS or with low permeability across the BBB and no central effects, and ii) the apparent Pe of the same drugs measured in animal models of the same species.…”

In vitro blood brain barrier models as a screening tool for colloidal drug delivery systems and other nanosystems

“…Although most research has focused on the development and characterisation of BBEC cultures, porcine brain endothelial cells (PBECs) are another convenient source with a sufficient number of BCECs to allow extensive testing. The use of primary cultures of PBECs as an in vitro permeability model of the BBB has been pioneered by Franke et al (1999), and more recent studies have also used co-cultured PBECs with astrocytes in order to improve the restrictiveness of the culture system (Kido et al, 2002;Zhang et al, 2006). Rat brain endothelial cells (RBECs) have also been described and may be relevant to facilitate in vitro/in vivo correlations if the rat is primarily used to generate in vivo data (Demeuse et al, 2002;Perriere et al, 2007).…”

“…It should be noted that, in absence of a three-dimensional model, performing permeability studies under stirred conditions on a two-dimensional co-culture model (reducing the influence of the unstirred water layer) better mimics the in vivo BBB environment and provides an intermediate response to the shear stress (Zhang et al, 2006).…”

“…Because there is strong evidence that glial cells dynamically interact with the BBB to regulate BBB cell properties, several groups have attempted to better recreate this brain environment. This goal has been achieved by co-culture of the endothelial cells together with either primary astrocytes or glial cells (Stanness et al, 1997;Zhang et al, 2006). Co-culture can be performed by growing the astrocytes on the bottom of a well in which a cell culture insert supporting endothelial cells is placed Dehouck et al, 1990;Perriere et al, 2005), or by seeding astrocytes directly on the bottom of the insert membrane (Megard et al, 2002) (Figure 5).…”

The Effect of ACTH4-10 synthetic on separated junction formation and CSF leukocyte number in LPS-induced meningitis

“…The mechanisms of separated junction formation relates to balance between contractile force of cytoskeleton and adhesive force in the junctional complex 5,16 . Under normal conditions, cerebral microvascular endothelial cells interacts closely one another 17 . In this study, we did not perform histological examination of normal brain tissue by ethical considerations, thus data distance between endothelial cells is not obtained.…”