A microfluidic platform for modeling drug transport and cell trafficking across the blood-brain barrier

 描述(申请人提供)：血脑屏障(BBB)是由微血管和毛细血管形成的紧密屏障，控制营养物质、液体、代谢产物和药物在血液和大脑之间的传递。血脑屏障功能受损与许多困扰大脑的主要病理有关，如阿尔茨海默病、多发性硬化症、帕金森氏病、艾滋病、中风和癌症的脑部表现。尽管神经治疗行业是制药行业中规模最大、增长最快的市场之一，但由于缺乏可靠地预测体内血脑屏障通透性的体外检测方法，目前进展受到阻碍。现有的模型都不能很好地复制血脑屏障的器官型微环境，在这种微环境中，脑内皮细胞(ECs)、周细胞(PC)和星形胶质细胞(ACS)排列在一个独特的结构中。这项拟议的工作利用诺蒂斯公司最近开发的芯片上器官技术，在一次性微流控芯片中创建3D组织微环境。该芯片设计能够整合活的、腔内灌流的微血管系统，使其适合研究屏障功能。引人注目的是，大量的初步数据表明，人脑ECs、PC和ACS有能力在Nortis芯片内自组装成类似BBB的架构。这些数据将被用来进一步开发小鼠和人类的血脑屏障模型，并最终实现商业化。第一阶段的目标是实现一种复制小鼠大脑关键血脑屏障功能的模型。将对小鼠模型进行开发和优化，以确保其生存能力、结构和功能。将测量紧密连接(TJ)蛋白和转运蛋白P-糖蛋白的表达，这是血脑屏障的一个重要功能特征。微血管通透性将通过荧光标记分子的灌流进行评估(目标1)。然后，该模型将被屏障调节化合物脂多糖(LPS)挑战，并评估TJ蛋白表达、分子通透性和白细胞跨内皮细胞迁移的相关变化(目标2)。在第二阶段，小鼠血脑屏障芯片将用于开发和鉴定特定的血脑屏障分析，如转铁蛋白受体转运体活性、内毒素对血脑屏障通透性的挑战以及刺激诱导的白细胞迁移(目标1)。成功的标准是Z‘≥0.2中的测试稳健性。第二阶段的目标2是开发一个人类血脑屏障模型。人体模型将进行优化，以概括BBB的关键结构和功能特征，包括TJ地层、渗透率和转运体活性。为了证明实用性，该模型将被内毒素、甘露醇和血管紧张素II治疗，并评估血脑屏障结构和功能的相关变化。这些化合物中的每一种都有临床意义，但作用机制不同。目标3是鉴定特定的人血脑屏障测定，并建立与临床数据的相关性。在这笔赠款的支持下开发的产品将极大地促进基础、翻译和临床神经科学研究的进展，并将极大地促进对许多毁灭性疾病的治疗。

 DESCRIPTION (provided by applicant): The blood-brain barrier (BBB) is a tight barrier formed by microvessels and capillaries that control the passage of nutrients, fluids, metabolic products, and drugs between the blood and the brain. Impaired function of the BBB is involved in a number of major pathologies afflicting the brain, such as Alzheimer's disease, multiple sclerosis, Parkinson's disease, brain manifestations of AIDS, stroke, and cancer. Although the neurotherapeutics sector is among the largest and fastest growing markets in the pharmaceutical industry, progress is currently impaired by the lack of in vitro assays that reliabl predict in vivo BBB permeability. None of the existing models adequately replicates the organotypic microenvironment of the BBB, in which brain endothelial cells (ECs), pericytes (PCs) and astrocytes (ACs) are arranged in a characteristic architecture. The proposed work utilizes organ-on-chip technology recently developed by Nortis, Inc. for creating 3D tissue microenvironments in disposable microfluidic chips. The chip design enables the integration of living, lumenally perfused microvasculature, making it suitable for studying barrier function. Strikingly, extensive preliminary data indicate that human brain ECs, PCs, and ACs have the capacity to self-assemble into a BBB-like architecture within the Nortis chip. This data will be leveraged to further develop and eventually commercialize BBB models of mouse and human. The objective of Phase I is to achieve a model that replicates critical BBB functions of the mouse brain. The mouse model will be developed and optimized for viability, structure, and function. Expression of tight-junction (TJ) proteins and the transporter P-glycoprotein, an important functional characteristic of the BBB, will be measured. Microvessel permeability will be assessed by perfusion with fluorescently labelled molecules (Aim 1). The model will then be challenged with the barrier-modulating compound lipopolysaccharide (LPS), and evaluated for associated changes in TJ protein expression, molecule permeability, and leukocyte transendothelial migration (Aim 2). During Phase II, the mouse BBB chip will be used to develop and qualify specific BBB assays, such as transferrin receptor transporter activity, BBB permeability challenge with LPS, and stimuli-induced leukocyte transmigration (Aim 1). Success criteria is an assay robustness of Z' ≥ 0.2. Aim 2 of Phase II is to develop a human BBB model. The human model will be optimized to recapitulate key structural and functional features of the BBB, including TJ formation, permeability, and transporter activity. To demonstrate utility, the model will be treated with LPS, mannitol, and angiotensin II and evaluated for associated changes in BBB structure and function. Each of these compounds has clinical relevance but acts by a different mechanism. Aim 3 is to qualify specific human BBB assays and establish relevance to clinical data. The products developed with support from this grant will significantly enhance progress in basic, translational, and clinical neuroscience research and will significantly advance therapy for numerous devastating diseases.