Blood-brain barrier modeling: challenges and perspectives

The blood-brain barrier (BBB) forms a highly selective interface between blood and brain. Extensive research efforts have completely changed our view of the BBB in the last years, from a static, impermeable barrier to a dynamic, highly regulated and specific cellular system. The BBB ultrastructurally consists of specialized brain microvascular endothelial cells (BMEC) characterized by low pinocytic activity, absent fenestrations and expression of specific and polarized transcellular transport systems. BMECs are interconnected by protein complexes consisting of tight and adherens junctions leading to high electrical resistance and low paracellular permeability. Attached pericytes and astrocytic end feet processes are critically involved in induction, sustaining and regulation of the BBB properties. Together with smooth muscle cells, neurons and circulating blood cells, they form the complex system of the neurovascular unit (NVU) (Neuwelt et al., 2011). The NVU controls the transition of fluids, molecules and cells between blood vessels and the central nervous system (CNS) integrating physiological and pathological signals such as metabolic needs, rheological disturbances and inflammatory stimuli. BBB dysfunction or disruption is a hallmark in the pathogenesis of primary or secondary (caused by systemic diseases) neurovascular, infectious, inflammatory or degenerative diseases of the CNS (Ruck et al., 2014). Hence, BBB research is of interest for multiple disciplines including neuroscience/neurology, (patho)physiology, pharmacology, internal medicine and many more. Given the importance of BBB research, there is a constantly increasing need for new and advanced model systems and techniques. Since human studies are restricted to post mortem investigations or imaging techniques such as magnetic resonance imaging (MRI) and positron emission tomography (PET) with limited resolution, nowadays most research on the BBB still involves laboratory animals. Vital dyes like Trypan Blue or Evans Blueto study in vivo permeability of the BBB have been used since the 19 century. Pharmacodynamic and pharmacokinetic properties of brain targeting drugs are still mostly studied with in vivo/in situ methods such as internal carotid perfusion or intracerebral dialysis fibers (Partridge, 1998). However, high financial and labour costs, limited transferability and overstraining complexity are in favour of parallel in vitro approaches. Advantages and disadvantages of the most important current and perspective in vitro models are discussed; an overview is presented in Figure 1 and Table 1. Isolated brain microvessels were the first approach of an in vitro BBB model (Joo, 1985). Microvessels are isolated from brain tissue by mechanic homogenization, enzymatic digestion and a variety of filtration and/or density gradient centrifugation techniques. This model system is easily accessible, well-established and maintains many structural and functional properties of the BBB. However, isolation of microvessels is labour intensive and difficult, viability of microvessels is often poor and it is not possible to study directional transport mechanisms or cellular transmigration. Isolation and cultivation techniques of brain-derived endothelial cells were developed in the early 1980s and remarkably facilitated in vitro BBB research. Cell-based approaches enable simplified, cost effective, highly flexible and easy controllable in vitro models of the BBB. Endothelial cells can be tapped from many different sources such as human or rodent brain tissue or cell-lineand stem-cell-derivatives (Lippmann et al., 2014). However, primary cells derived from humans or genetically modified animals are of limited availability, critically affecting costand time-effectiveness. On the other hand, endothelial cell lines cannot fully substitute for primary cells as important cell compromising translation of results properties are altered to the in vivo situation. Moreover, the isolation of endothelial cells from transgenic animals or from patients allows for the reproduction and investigation of mechanisms involved in initiation and/or progression of several CNS diseases. An advanced understanding of these mechanisms is essential for the development of new therapeutic strategies. Transwell-systems are the most common and widely used cell-based in vitro models of the BBB.