InVitro BBB Models Cells and Devices

A minimal approach to an in-vitro BBB model focuses mainly on brain microvascular ECs (see Table 1.3). For the past three decades, capillaries have been prepared from the brains of various species of embryonic or adult animals. Brain ECs have been isolated and primary ECs were cultured in vitro. The disadvantages of using primary cultures include:

• the extensive use of animals,

• tedious, complicated protocols for cell isolation,

• slow cell proliferation rates,

• the limitation in passaging these cells.

Cultured primary ECs quickly (within a few population doublings) lose the majority of their barrier-related characteristics, such as expression levels of transporters. Similarly, the transendothelial electrical resistance (TEER) is high immediately after isolation but declines significantly in culture [180]. Endothelial cell lines, which are obtained from tumors or immortalized upon infection with viruses or viral constructs, and can be propagated for many generations, appear to be more convenient for generating in-vitro BBB models. However, as inferred from electrical resistance and permeability measurements, transformed EC-based BBB models exhibit barrier properties inferior to BBB models employing primary brain capillary ECs. Moreover, due to the high variability between different cell lines, each cell line-based BBB model must be characterized extensively [180-182].

High TEER values are considered to be a physiological marker of an intact, functional BBB indicative of the organotypic differentiation state of brain ECs. Remarkably, some ECs - irrespective of their origin - have the potential to differentiate into a BBB phenotype in culture. This plasticity depends on the culture conditions [183]. Thus, a systematic search for differentiating growth factors should be performed in order to optimize the barrier properties of ECs.

Two major cell banks (; provide some of the currently available commercial EC lines for BBB models. Primary capillary ECs are mostly investigator-generated by isolation and purification from brain capillaries of a variety of species (e.g., porcine, bovine, rat, mouse, monkey, human). To date, no uniform protocols exist for the isolation and culture of these primary cultures. Although comparisons between different laboratories are difficult, it seems clear that primary ECs show more prominent barrier properties (see Table 1.3), exhibiting higher TEER values and lower paracellular permeability. However, the role of TEER as a predictor of the tightness of an EC monolayer is rather questionable and uninformative with regard to the paracellular permeability [184].

More sophisticated in-vitro BBB models must include, in addition to ECs, also astrocytes. Astrocytes can be obtained either from primary cultures of dissociated brain cells, or they exist as established cell lines. In co-culture with ECs, astrocytes, cell lines and primary cultures, are each capable of inducing endothelial-monolayer tightening/differentiation toward the BBB phenotype by secreting as yet unidentified growth factors, and establishing cell-cell contact-based interactions of the glial feet extensions with the ECs [185-187]. The most commonly used astrocytic cell line is C6-glioma, a cell line isolated from a rat glioma (Table 1.3). However, a major disadvantage of C6 glioma cells - as well as of other astrocytomas - is their ability to release VEGF (also called vascular permeability factor, VPF), which counteracts the endothelial BBB barrier functions in the in-vitro co-culture model [188-190].

BBB experiments in vitro are most frequently conducted with Transwell™ or similar filter systems. These are special tissue culture plates that enable separation of the ECs from glia by a thin, porous, polymeric membrane. This system was originally developed for modeling gastrointestinal permeability [191], and later adopted for other in-vitro permeability studies, including BBB models. A simple, static model of the BBB is generated by growing a confluent endothelial monolayer on top of the insert, while on the apposing side glial cells are grown (Fig. 1.7B). Several manufacturers produce transwells for pharmacological models; the most useful for BBB studies are filters made of transparent, polyethylene terephthalate (PET) with a pore size of 0.4 or 1.0 (xm. These inserts allow for unimpaired exchange of small molecular nutrients and test compounds with a molecular weight < 1000 Da. At the same time, cells seeded on both sides of these inserts can interact with each other through direct cell-cell contacts via "end-feet" - that is, cell extensions penetrating through the pores (Fig. 1.7C and insert). As a first approximation of the BBB, this configuration mimics compartmentalization between the apical side (blood vessel lumen) and basolateral (brain parenchyma, for example, glia and neurons).

The ECs respond in different ways to the polymeric substrates on which they are grown [171], and consequently the type of the filter may influence endothelial behavior. This issue is extremely important when generating in-vitro BBB models. In many experimental situations, it is important to visually examine the interactions of the endothelial monolayer, and therefore only completely transparent filters, such as PET, should be chosen. In addition, the pore diameter of the filters has important implications for establishing cell-cell contacts in co-cultures [192]. Finally, the cost of each insert must also be taken into consideration, especially for large-scale permeability screenings.

The TEER across intact endothelial monolayers is a simple, fast, reproducible, and nondestructive measurement of monolayer tightness and drug-induced changes therein. The electrical resistance is generated because of the restriction by the tight junctions of the flow of small ions through the monolayer. There is a reciprocal relationship between electrical resistance and ion flow; for instance, the tighter the monolayer, the lower the flow, and, hence, the higher the electrical resistance across the BBB. In vivo, TEER values can exceed 2 kO X cm- , whereas in most in-vitro models, TEER values of < 100 Q cm- are obtained.

In addition to the TEER, the permeability ofdiverse compounds through cellular monolayers, and in particular in-vitro BBB models, can also be assessed from their permeability coefficient, P [193]. Unfortunately, for a given EC type the P-values seem to depend as much on the nature of the filters used, as on the physico-chemical properties of the compounds investigated. The commonly accepted assumption has been that polymeric filters are inert and have no influence on the permeability of in-vitro BBB models. This assumption is inaccurate, however. The studies by Yu and Sinko [194] and Lauer et al. [195] indicate that the barrier properties of EC lines might change according to the composition of the filters in the transwell systems. Therefore, novel biocompatible supports/inserts for in-vitro BBB models need to be developed. Likewise, the protocol for designing and testing in-vitro BBB models should be strictly defined and standardized between individual laboratories, in order to facilitate stringent pharmacological studies.

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