Development of a BBB Model to Study Transendothelial Cell Migration

描述（由申请人提供）：越来越多的证据表明，全身性炎症和血脑屏障（BBB）（其成为过度反应或误导的免疫细胞的靶点，这些免疫细胞决定BBB衰竭和免疫外渗到脑实质中）参与了神经系统疾病（如脑膜炎、炎症、阿尔茨海默病和多发性硬化症）的发病机制。因此，了解白细胞运输到大脑的机制可能会为如何调节病理性免疫反应或增强神经炎性疾病中的宿主保护机制提供见解。成功解决这一关键问题和开发新型药物治疗的关键是使用能够详细再现BBB生理学及其对炎症过程的功能反应的人工系统。到目前为止，我们已经开发了一种基于微孔中空纤维技术的基于流动的人工共培养系统（DIV-BBB），该系统能够再现准生理环境，其中内皮细胞和星形胶质细胞建立功能性BBB。这种BBB模型已被证明是密切模仿的特点和功能特性在体内。然而，来自该实验室和其他实验室的大量证据表明，研究BBB在神经系统疾病中的作用的该模型的主要限制是由于跨毛细血管孔的直径小（0.2- 0.55 m）而缺乏跨内皮细胞运输。此外，由于动态体外血脑屏障模型（DIV-BBB）更准确地反映了包括血脑屏障的毛细血管的性质，因此尚不完全清楚该系统是否适用于研究脑中可能发生在毛细血管后段（微静脉）的白细胞外渗。因此，为了解决这个关键问题，我们还建议原型和验证后毛细血管（DIV-Venules）接口，这将被添加到DIV-BBB开发的第一个体外毛细血管-微静脉模型的脑血管。为此，该1期STTR提案的目的如下：具体目的1：建立脑血管系统的新的动态体外毛细血管-小静脉模型原型，该模型允许白色血细胞（WBC）从血管外渗到系统的脑实质（脑）侧。为此，我们将研究在为血管细胞生长提供结构支持的人工中空纤维中制造大直径孔（2-4 5 m）的三种方法，并且我们将确定大规模生产这些改性人工毛细管的最具成本效益的方法。具体目标2：评价动态体外毛细血管-微静脉模型，并根据平行Transwell模型验证系统。a）测量药代动力学（例如，对高极性分子的细胞旁渗透性）、细胞活力和DIV-BB B和DIV-小静脉模块的其它独特的血管特性; B）通过评估THP-1细胞（人单核细胞系）在毛细血管和小静脉模块的脑区室中响应于腔外趋化因子的外渗，并确定THP-1迁移的外渗模式（毛细血管对小静脉）。这种新的体外脑毛细血管-微静脉模型的生理反应将与平行的Transwell系统进行比较，Transwell系统通常被认为是脑血管研究的金标准。 公共卫生相关性：了解人类神经系统疾病需要同时研究各种细胞类型（例如，神经元、内皮、星形胶质细胞、白色血细胞等）以及流体相因子（粘附分子、细胞因子、促炎因子、血管内剪切力等）。血脑屏障（BBB）证实了这种方法对神经科学的重要性。BBB功能的丧失在许多中枢神经系统（CNS）疾病的发病机制中起着关键作用。在本项目中，我们将设计一种新的动态体外血脑屏障模型（nDIV-BBB）并制作原型，该模型的孔径允许白色血细胞（WBC）从内皮细胞的基底表面渗出到测试卡片的细胞外空间。此外，我们将研究在中空纤维中制造孔的三种方法，以确定大规模生产墨盒的最具成本效益的方法。我们已经表明，DIV-BBB模型比二维（平板）模型更准确地表示体内血脑屏障的特征。然而，DIV-BBB模型的当前孔径不允许需要单核细胞外渗屏障的炎症研究。此外，我们将通过评估细胞生长和BBB活力以及具有低细胞旁通透性的紧密屏障的形成来验证这些改进的DIV-BBB模型的可行性。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: 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.