A dynamic in vitro BBB model for the study of immune cell trafficking into the central nervous system

Abstract

Although there is significant evidence correlating overreacting or perhaps misguided immune cells and the blood-brain barrier (BBB) with the pathogenesis of neuroinflammatory diseases, the mechanisms by which they enter the brain are largely unknown. For this purpose, we revised our humanized dynamic in vitro BBB model (DIV-BBBr) to incorporate modified hollow fibers that now feature transmural microholes (2 to 4 μm Ø) allowing for the transendothelial trafficking of immune cells. As with the original model, this new DIV-BBBr reproduces most of the physiological characteristics of the BBB in vivo. Measurements of transendothelial electrical resistance (TEER), sucrose permeability, and BBB integrity during reversible osmotic disruption with mannitol (1.6 mol/L) showed that the microholes do not hamper the formation of a tight functional barrier. The in vivo rank permeability order of sucrose, phenytoin, and diazepam was successfully reproduced in vitro. Flow cessation followed by reperfusion (Fc/Rp) in the presence of circulating monocytes caused a biphasic BBB opening paralleled by a significant increase of proinflammatory cytokines and activated matrix metalloproteinases. We also observed abluminal extravasation of monocytes but only when the BBB was breached. In conclusion, the DIV-BBBr represents the most realistic in vitro system to study the immune cell trafficking across the BBB.

Figure 1

Cross-sectional view of artificial capillaries used in our model. Endothelial cells (ECs) cocultured with abluminal astrocytes in the dynamic in vitro blood–brain barrier (DIV-BBB) under flow condition develop a phenotype similar to that of the brain microvascular EC in situ.

Figure 2

Schematic representation of the dynamic in vitro blood–brain barrier (DIV-BBB) model. (A) A bundle of porous polypropylene hollow fibers is suspended in the DIV-BBB chamber. The hollow fibers are in continuity with a medium source through a flow path consisting of gas-permeable silicon tubing. Note that the original hollow fibers have been modified to accommodate transcapillary microholes, which now make the artificial capillary permissive for cell extravasation (B). ECS, extraluminal space; WBC, white blood cells.

Figure 3

Development of a functional blood–brain barrier (BBB) in the new dynamic in vitro (DIV)-BBB system. (A) The presence of larger transcapillary pores in the revised DIV-BBB does not impact the process of formation of the integrity of the BBB measured by transendothelial electrical resistance. The asterisk indicates a non-statistically significant difference (P>0.05) during the early stage of BBB formation in the new versus the conventional DIV-BBB. Note the microholes manufactured in the hollow fibers under transmitted light microscopy. (B) Permeability measurements to sucrose, phenytoin, and diazepam reflect the formation of a BBB capable of discriminating between solutes with different lipophilicity. Please note that the counts per minute (c.p.m.) versus time curves reflect the relative amount of solutes (expressed in c.p.m.) in the lumen and in the extraluminal space (ECS) at a given time point. (C) The dashed line indicates the idealized relationship if the data in vivo were identical to in vitro. Note how permeability obtained in the DIV-BBB closely mimic the in vivo scenario. TEER, transendothelial electrical resistance.

Figure 4

Functional characterization of the dynamic in vitro blood–brain barrier (DIV-BBB). (A) Under dynamic (flow) culture conditions, the ratio between glucose consumption and lactate production is ≈1. This indicates increased propensity toward an aerobic metabolic pattern, which is a well-established indicator of the formation of a functional BBB (Santaguida et al, 2006). The asterisk indicates a statistically significant difference (P<0.05) versus parallel systems established under static conditions. (B) Hyperosmolar opening of the BBB in DIV models was assessed by real-time measurements of transendothelial electrical resistance (TEER). Similar to what was observed in vivo, the transient nature of the BBB opening is indicative of the formation of an in vitro BBB that closely mimic the physiological response of that in situ.

Figure 5

The new dynamic in vitro blood–brain barrier (DIV-BBB) model allows for the extravasation of monocytes when the BBB is breached. (A, left side) Transendothelial electrical resistance (TEER) measurements show the longitudinal progression of BBB formation in the conventional and new DIV-BBB used for the flow cessation reperfusion experiments. (A, right side) Flow cessation reperfusion (Fc/Rp, flow blockade for 1 hour) in the presence of circulating white blood cells causes a biphasic opening of the BBB. This loss of BBB integrity occurs earlier (1 hour) and is prolonged (2 hours) in the new DIV system where extravasation of cells is possible. Note that extravasation of monocytes into the abluminal (parenchymal) space occurs only when the DIV module featuring larger transcapillary pores was subjected to Fc/Rp (B). Monocytes, later identified by fluorescence activated cell sorting (FACS), are indicated with asterisk. No extravasation is observed in the traditional DIV-BBB model. These results suggest the possibility that an abluminal macrophagic action of activated monocytes may contribute to extend onset and duration of blood–brain barrier disruption (BBBD). ECS, extraluminal space; WBC, white blood cells.

Figure 6

Effects of flow cessation reperfusion on blood–brain barrier (BBB) integrity are mediated by cytokines and matrix metalloproteinases (MMPs). (A) The loss of BBB integrity was paralleled by the release of the proinflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor α (TNF-α) as well as the release (B) and activation of MMP-2 and -9 (C). Note also that the activity level of MMP-9 measured in media samples from the new dynamic in vitro (DIV)-BBB system were significantly higher than that observed in the conventional model. The asterisk indicates a statistically significant difference (P<0.05) versus controls. The double asterisk in panel C indicates a statistically significant difference (P<0.05) between 2 and 12 hours postreperfusion corresponding to the occurrence of the second opening. The triple asterisk indicates a statistically significant difference (P<0.05) between MMP-9 activity assessed in the new DIV-BBB system versus the conventional model.

Figure 7

Schematic representation of capillary and postcapillary structure of the brain circulatory system in comparison to that of the dynamic in vitro blood–brain barrier (DIV-BBB). The figure shows a schematic representation of the cross-section of brain precapillary (A) and capillary (B) segments in situ and that of a hollow fiber used to manufacture the DIV-BBB (C). Note that the overall thickness of the hollow fibers (150 μm), which provide the support for endothelial cells and abluminal astrocytes is significantly larger than the basal lamina present at the endothelial–glia interface in situ. However, it is likely that the thickness of the hollow fiber interface is significantly reduced within the area of each microhole. Note also that the Virchow–Robin spaces that surround the precapillary segments are replaced by the hollow fiber wall, where the only empty space is represented by the transmural pores and microholes.