Increasing evidence indicates that systemic inflammation and the blood-brain barrier (BBB), which becomes the target of overreacting or misguided immune cells that determine BBB failure and immune extravasations into the brain parenchyma, are involved in the pathogenesis of neurological diseases such as meningitis, inflammation, Alzheimer's disease, and multiple sclerosis. Therefore understanding the mechanisms of leukocyte trafficking into the brain might provide insights into how to modulate pathologic immune responses or enhance host protective mechanisms in neuroinflammatory diseases. Essential for the success of this critical issue and for the development of novel pharmacological treatments is the use of artificial systems capable to reproduce in detail the physiology of the BBB and its functional response to the inflammatory processes. To date, we have developed a flow-based artificial co-culture system (DIV-BBB) based on microporous hollow fiber technology that is capable to reproduce a quasi-physiological environment where endothelial cells and astrocyte establish a functional BBB. This BBB model has been shown to closely mimic the characteristics and functional properties of in vivo. However, a significant body of evidence from this and other laboratories suggests that the main limitation of this model to study the role of the BBB in neurological diseases is lack of transendothelial cell trafficking due to the small diameter of the transcapillary pores (0.2-0.55m). Moreover, because the dynamic in vitro BBB model (DIV-BBB) more accurately reflects the properties of capillaries comprising the BBB;it is not entirely clear whether this system is appropriate for studying leukocyte extravasation in the brain, which is likely to occur at the post-capillary segment (venules). Therefore, to address this critical issue we also propose to prototype and validate a post-capillary (DIV-Venules) interface, which will be added to the DIV-BBB to develop the first in vitro capillary-venules model of the brain cerebrovasculature. To this end, the aims of this Phase 1 STTR proposal are the following:
Specific Aim 1 : To prototype a new dynamic in vitro capillary-venules model of the brain cerebrovasculature that is permissive for the extravasation of white blood cells (WBC) from vascular into the parenchimal (brain) side of the system. To this end, we will investigate three methods of manufacturing large diameter holes (2-4 5m) in the artificial hollow fibers that provide the structural support for vascular cell growth and we will determine the most cost effective way to mass-produce these modified artificial capillaries.
Specific Aim 2 : To evaluate the dynamic in vitro capillary-venules model and validate the system against parallel Transwell models. This will be assessed by: a) Measuring the pharmacokinetic (e.g., paracellular permeability to high polar molecules), cell viability, and other distinctive vascular properties of the DIV-BBB and the DIV-Venules modules;b) By assessing the extravasation of THP-1 cells (human monocytic cell line) in the brain compartments of the capillaries and venules modules in response to abluminal chemokines and to determine the patterns of extravasation (capillary versus venules) of THP-1 migration. The physiological response of this new in vitro brain capillary-venules model will be compared against parallel Transwell systems, which are generally considered the gold standard in cerebrovascular research.
Understanding human neurological diseases requires simultaneous studies of various cell types (e.g., neurons, endothelium, astrocytes, white blood cells, etc.) as well as fluid phase factors (adhesion molecules, cytokines, pro-inflammatory factors, intravascular shearing forces, etc). The blood-brain barrier (BBB) exemplifies the importance of this approach to neuroscience. Loss of BBB function plays a pivotal role in the pathogenesis of many diseases of the central nervous system (CNS). In this project, we will design and prototype a new dynamic in vitro blood-brain barrier model (nDIV-BBB) with a pore size that allows white blood cells (WBC) to extravasate from the basal surface of the endothelial cells into the extracellular space of the cartridge. In addition, we will investigate three methods of manufacturing holes in the hollow fiber to determine the most cost effective way to mass-produce the cartridges. We have shown that the DIV-BBB model more accurately represents the characteristics of an in vivo blood- brain barrier than the two-dimensional (flat plate) models. However, the current pore size of the DIV-BBB model does not allow for inflammation studies that require monocytes to extravasate the barrier. In addition, we will validate the feasibility of these improved DIV-BBB models by assessing the cell growth and BBB viability as well as the formation of a tight barrier with low paracellular permeability.
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