BBB InVitro Models From First to Third Generation the Biological Approach

The first generation of in-vitro models of the BBB was represented by monocultures ofECs, isolated from diverse vessels and different animal species. By culturing ECs on top ofvarious transwell inserts, this approach was useful to establish the growth requirements ofthe cells on the inserts. Apparently, confluent 2D monolayers were then used to measure TEER values and to investigate the permeability of low and high molecular-weight markers (see Table 1.3). The major conclusion from these studies was that such in-vitro systems, comprised of only EC monolayers, could only partially mimic the properties of the in-vivo BBB as the permeabilities were too high and the TEER values too low. Therefore, the pharmacological relevance of these models is rather limited.

The second generation of in-vitro models of the BBB (Table 1.3) is comprised of co-cultures of ECs (primary isolates or immortalized ECs lines derived from bovine, porcine, rodent or human brain capillaries) that were seeded on the top of the transwell inserts, and of rodent or human astrocytes, primary or transformed (C6 glioma), grown on the other side of the insert [196]. While the ECs, immortalized with SV40 or polyoma virus large T-antigen or adenovirus E1A genes, continued to express a variety of endothelial markers and responded to astroglial factors, most of these models still exhibited low TEER values and relatively high paracellular permeability to small molecules [196], thus limiting their use for pharmaceutical studies. However, these studies were very important in defining the minimal size of the transwell pores (> 0.8 (im diameter) necessary to allow penetration of glial feet onto the endothelial monolayer, and also to demonstrate the presence and activity of a variety of transporters and tight junction proteins in BBB-type ECs [197]. Furthermore, these experiments confirmed that C6 glioma cells are ill-suited as substitutes for primary glial cells [198], and also led to the conclusion that the specific astrocytic factors involved in inducing the BBB phenotype in ECs remain to be discovered [199].

The third generation of in-vitro BBB models (Table 1.3) begins to address the contribution of other BBB cellular components such as neurons and pericytes on the organization and function of brain endothelium, and the multidirectional interactions between these components. Despite their localization close to capillaries and tight interactions with glia, very little is known about the contribution of neurons to the properties of the BBB [200]. For example, co-cultures of RBE4 ECs with rat primary neurons indicated a reduction in the transmonolayer dopamine flux [193]. In a recent, unique study, a three-cell type in-vitro BBB was generated by culturing primary brain capillary ECs on top of the transwell insert, while neurons and astrocytes from the same animal were grown on the opposite side. This configuration synergistically enhanced the expression of the tight-junction protein occludin, while paradoxically showing an increase in paracellular permeability [201]. Pericytes have a fundamental role in vivo to stabilize all capillaries, including those in the brain. In a recent in-vitro BBB model, it appears however that pericytes reduce endothelial paracellular permeability [202], most probably by continuous production of TGF-P [203].

The information obtained with these three generations of in-vitro BBB models, while providing important data on fundamental properties of such a model, also highlights the limitations ofcurrent models. This lack of a valid, high-fidelity BBB model highlights the urgent need for innovative approaches towards engineering pharmaceutically relevant novel in-vitro BBB models using an integrated concept. Such a concept should include modern biomaterials and biotechnological approaches in conjunction with a heightened understanding of the specific and unique roles that each of the cellular component plays in establishing and maintaining the BBB.

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