Development of a BBB Model to Study Transendothelial Cell Migration

描述(申请人提供)：越来越多的证据表明，全身炎症和血脑屏障(BBB)，成为反应过度或被误导的免疫细胞的目标，决定了BBB的失效和免疫渗入脑实质，参与了脑膜炎、炎症、阿尔茨海默病和多发性硬化症等神经系统疾病的发病机制。因此，了解白细胞进入大脑的机制可能会为如何在神经炎症性疾病中调节病理性免疫反应或增强宿主保护机制提供见解。这一关键问题的成功和开发新的药物治疗方法的关键是使用能够详细再现血脑屏障的生理及其对炎症过程的功能反应的人工系统。到目前为止，我们已经开发出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)，能够再现内皮细胞和星形胶质细胞建立功能性BBB的准生理环境。这种血脑屏障模型已被证明非常接近体内的特征和功能特性。然而，来自该实验室和其他实验室的大量证据表明，该模型研究血脑屏障在神经系统疾病中的作用的主要限制是由于跨毛细血管的小直径(0.2-0.55m)而缺乏跨内皮细胞的运输。此外，由于动态体外血脑屏障模型(DIV-BBB)更准确地反映了组成血脑屏障的毛细血管的特性；目前尚不完全清楚该系统是否适合于研究脑内白细胞外溢，这可能发生在毛细血管后段(小静脉)。因此，为了解决这一关键问题，我们还提议制作并验证毛细血管后接口(DIV-Venules)，该接口将被添加到DIV-BBB中，以开发第一个体外脑血管模型。为此，此第一阶段STTR方案的目标如下：具体目标1：建立一种新的脑血管体外动态毛细血管-微静脉模型，允许白细胞(WBC)从血管渗入系统的实质(脑)侧。为此，我们将研究三种在人造中空纤维中制造大直径孔(2-4.5米)的方法，为血管细胞的生长提供结构支持，并确定批量生产这些改性人造毛细血管的最具成本效益的方法。具体目的2：评估体外动态毛细血管-小静脉模型，并对照平行Transwell模型验证该系统。将通过以下方式进行评估：a)通过测量DIV-BBB和DIV-小静脉模块的药代动力学(例如，细胞旁对高极性分子的通透性)、细胞活力和其他独特的血管特性；b)通过评估THP-1细胞(人单核细胞系)对开拓性趋化因子的反应在脑室毛细血管和小静脉模块中的外渗情况，并确定THP-1迁移的外渗模式(毛细血管和小静脉)。这种新的体外脑毛细血管-小静脉模型的生理反应将与平行的Transwell系统进行比较，后者通常被认为是脑血管研究的金标准。

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.