Development of a BBB Model to Study Transendothelial Cell Migration

开发 BBB 模型来研究跨内皮细胞迁移

• 批准号: 7998687
• 负责人: DAMIR JANIGRO
• 金额: $ 9.1万
• 依托单位: FLOCEL, INC.
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-27 至 2012-06-26
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7998687
• 关键词: Address; Alzheimer' s Disease; Architecture; Astrocytes; Blood - brain barrier anatomy; Blood Vessels; Blood capillaries; Brain; Caliber; Cell Adhesion Molecules; Cell Line; Cell Survival; Cells; Central Nervous System Diseases; Cerebrovascular system; Characteristics; Chemotactic Factors; Coculture Techniques; Data; Development; Disease; Drug Kinetics; Endothelial Cells; Endothelium; Environment; Extracellular Space; Extravasation; Failure; Fiber; Future; Gold; Human; Immune; Immune response; In Vitro; Inflammation; Inflammatory; Laboratories; Leukocyte Trafficking; Leukocytes; Liquid substance; Lymphocyte Activation; Measures; Meningitis; Methods; Modeling; Molecular; Multiple Sclerosis; Neuraxis; Neurons; Neurosciences; Pathogenesis; Pathologic; Pattern; Perfusion; Pericytes; Permeability; Pharmacological Treatment; Phase; Phenotype; Physiological; Physiology; Play; Process; Property; Research; Role; Side; Signal Transduction; Small Business Technology Transfer Research; Structure; Surface; System; Technology; Validation; Vascular Endothelium; Vascular Smooth Muscle; Venous; base; capillary; cell growth; cell motility; cell type; cerebrovascular; chemokine; cost effective; cytokine; design; hemodynamics; improved; in vivo; insight; manufacturing process; migration; monocyte; nervous system disorder; novel; prototype; public health relevance; response; shear stress; success; trafficking; two-dimensional; venule

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.

描述（由申请人提供）：越来越多的证据表明，系统性炎症和血脑屏障（BBB）成为确定BBB衰竭和免疫渗入脑部实质性的过度反应或误导的免疫细胞的靶标，与脑实质有关神经系统疾病，例如脑膜炎，炎症，阿尔茨海默氏病和多发性硬化症。因此，了解白细胞运输到大脑的机制可能会提供有关如何调节病理免疫反应或增强神经炎症性疾病中宿主保护机制的见解。对于这个关键问题的成功和新型药理治疗的发展至关重要的是，使用人工系统详细介绍了BBB的生理及其对炎症过程的功能反应。迄今为止，我们已经开发了一种基于微孔空心纤维技术的基于流动的人工共培养系统（DIV-BBB），该系统能够重现一个准生理环境，其中内皮细胞和星形胶质细胞建立功能性BBB。该BBB模型已被证明可以密切模仿体内的特性和功能特性。但是，该模型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主要局限性是由于经毛孔的直径较小而缺乏跨内皮细胞运输（0.2-0.555m ）。此外，由于动态体外BBB模型（DIV-BBB）更准确地反映了包含BBB的毛细血管的性能；目前尚不清楚该系统是否适合研究大脑中的白细胞渗出，这很可能发生在毛细血管后段（venules）。因此，为了解决这个关键问题，我们还建议原型并验证毛细血管后（分形式）界面，该界面将添加到DIV-BBB中，以开发大脑大脑大脑大脑脑管的第一个体外毛细血管模型。为此，此阶段1 STTR提案的目的如下：特定目的1：针对脑大脑大脑大脑毛细管模型的新动态性动态性毛细血管模型，该模型允许渗出白细胞（WBC）从血管中渗出进入系统的极地（大脑）侧。为此，我们将研究三种在人工空心纤维中生产大直径孔（2-4 5m）的方法，这些方法为血管细胞生长提供了结构支持，我们将确定最具成本效益的方法来批量生产这些改良的人工化。毛细血管。特定目的2：评估动态体外毛细管模型，并针对平行式Transwell模型验证系统。这将通过：a）测量药代动力学（例如，对高极性分子的细胞细胞渗透性），细胞活力以及DIV-BBB和分层模块的其他独特血管特性； b）通过评估毛细血管和静脉模块中Thp-1细胞（人类单核细胞系）的渗出，以响应于阿布林纳尔趋化因子，并确定thp-1迁移的溢出模式（毛细血管与静脉）的模式。这种新的体外脑毛细血管结膜模型的生理反应将与平行的跨层系统进行比较，这些毛细管系统通常被认为是脑血管研究中的黄金标准。 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，白细胞等）以及液相因子（粘附分子，细胞因子，炎症性因素，促脑膜内剪切因子部队等）。血脑屏障（BBB）体现了这种方法对神经科学的重要性。 BBB功能的丧失在中枢神经系统（CNS）的许多疾病的发病机理中起关键作用。在这个项目中，我们将设计和原型一种新的动态体外血脑屏障模型（NDIV-BBB），其孔径允许白细胞（WBC）从内皮细胞的基础表面渗入进入细胞外空间墨盒。此外，我们将研究三种在空心纤维中制造孔的方法，以确定大量生产墨盒的最具成本效益的方法。我们已经表明，与二维（平板）模型相比，DIV-BBB模型更准确地表示体内血液屏障的特征。但是，DIV-BBB模型的当前孔径不允许进行炎症研究，而炎症研究需要单核细胞来外观。此外，我们将通过评估细胞的生长和BBB生存能力以及形成低细胞细胞通透性的紧密屏障来验证这些改善的DIV-BBB模型的可行性。