Tridimensional Dynamic Models of the Blood Brain Barrier Flow Based

Both short- and long-term changes occur in cerebral arterioles in response to intraluminal flow [5]. Endothelial cells in vivo are continuously exposed to shear stress generated by blood flow. A new in vitro model of the BBB has recently been developed and is characterized by a tridimensional, pronectin-coated hollow fiber structure that enables cocul-turing of ECs with glia (Figure 2) [6]. In the hollow-fiber apparatus, ECs are seeded intraluminally and are exposed to

Hollow Fibre Coculture

Figure 2 Tridimensional in vitro model of the BBB: Endothelial cells are seeded intraluminally in a pronectin-coated hollow fiber, while astrocytes are cultured extraluminally. Left panel: Hollow fiber section (L, lumen; ECS, extracellular space); right panel: schematic representation. (see color insert)

Figure 2 Tridimensional in vitro model of the BBB: Endothelial cells are seeded intraluminally in a pronectin-coated hollow fiber, while astrocytes are cultured extraluminally. Left panel: Hollow fiber section (L, lumen; ECS, extracellular space); right panel: schematic representation. (see color insert)

flow. Under these conditions, endothelial cells develop a morphology that closely resembles the endothelial pheno-type in situ, demonstrating that endothelial cells that are grown under flow conditions develop a greater differentiation than conventional culture. We reported the induction of BBB properties in endothelial cells grown in hollow fibers in the presence of extraluminally seeded glia; this induction of a BBB-specific phenotype included low permeability to intraluminal potassium, negligible extravasation of proteins, and the expression of a BBB-specific glucose transporter. In addition, coculturing of EC with glia affected the overall morphology of these cells and induced the expression of BBB-specific ion channels [6, 7].

The model system used for these studies resulted from a modification of a traditional cell culture system normally used for expansion of hybridoma cells, and the general design of the hollow-fiber apparatus was derived from attempts toward the development of a "cell factory." Cell culture on hollow fibers has been extensively exploited for mass production of rare cell types, antibody production, and modeling of organlike structures such as the blood-brain barrier. Ott et al. [8] used a hollow fiber cell culture apparatus for studies of flow-mediated effects on endothelial cell growth, while Stanness and her colleagues [4] further developed this system by performing permutations of intra- and extraluminal cell growth to study the effects of glia on endothelial cells.

The dynamic in vitro BBB model (DIV-BBB) is constituted by a plastic support containing a variable number of artificial capillaries (approximately 300 mm cross diameter with a lumen of approximately 75 mm). These capillaries bear 0.5-|mm transcapillary pores that allow free diffusion of solutes from the extraluminal compartment to the intralumi-nal space and vice versa. The capillaries are intraluminally perfused at various shear stress rates by pulsatile flow (Figure 3). It was shown that induction of BBB-like characteristics occurs following prolonged coculture of glia and bovine

Figure 3 Schematic representation of the tridimensional dynamic in vitro model (DIV-BBB); cells are injected into the system through the loading ports and the flow is maintained by a pump. (see color insert)

aortic endothelial cells and that glia can induce the expression of BBB-specific ion channel proteins in non-BBB endothelial cells [6]. Interestingly, genetically altered astro-cytes lacking intermediate filaments were not capable of these induction properties [9].

Several improvements in model design have allowed for better understanding and study of the central nervous system and neurological diseases. This model in particular has several advantages compared to mono- or bidimensional models, such as closely mimicking the conditions of pre-and postcapillary vessels in vivo, and allowing for experimental manipulations and reutilization of the device. In this model, high transendothelial electrical resistance (TEER), low permeability to sucrose, and stereoselective transport also prevail. It is also possible to perform long-term studies (months), and the presence of drug extrusion mechanisms allows for meaningful drug permeation studies. However, there are obvious limitations to this model, including, but not limited to, lack of blood cells, no intraluminal pressure changes, and a lack of neuronal influences. Some of these issues have been addressed recently by Krizanac-Bengez and co-workers [10].

Table II Advantages and Disadvantages of Cell Lines versus Primary Human Cultures.

Cell lines

Primary human cultures

Pros

Cost effective

Variety of etiologies available

Easy to use

Not manipulated

Readily available

Don't divide uncontrollably

Cons

Some conditions/diseases

Obtaining human specimens

may be unable to be

Skills

mimicked by these cells

Number of cells isolated

May become

hyperproliferative or

"immortalized"

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