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通过。血脑屏障功能受损与许多影响大脑的主要疾病有关，例如阿尔茨海默病、多发性硬化症、帕金森病、艾滋病的大脑表现、中风和癌症。尽管神经治疗领域是制药行业最大且增长最快的市场之一，但由于缺乏可靠预测体内血脑屏障渗透性的体外测定，目前的进展受到损害。现有的模型都没有充分复制 BBB 的器官型微环境，其中脑内皮细胞 (EC)、周细胞 (PC) 和星形胶质细胞 (AC) 以特征结构排列。拟议的工作利用 Nortis, Inc. 最近开发的器官芯片技术在一次性微流控芯片中创建 3D 组织微环境。该芯片设计能够集成活体、管腔灌注的微血管系统，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 EC、PC 和 AC 能够在 Nortis 芯片内自组装成类似 BBB 的架构。这些数据将用于进一步开发小鼠和人类的 BBB 模型并最终将其商业化。第一阶段的目标是建立一个复制小鼠大脑关键 BBB 功能的模型。小鼠模型将在活力、结构和功能方面进行开发和优化。将测量紧密连接 (TJ) 蛋白和转运蛋白 P-糖蛋白（BBB 的重要功能特征）的表达。将通过荧光标记分子灌注来评估微血管通透性（目标 1）。然后，该模型将受到屏障调节复合脂多糖 (LPS) 的挑战，并评估 TJ 蛋白表达、分子通透性和白细胞跨内皮迁移的相关变化（目标 2）。在第二阶段，小鼠 BBB 芯片将用于开发和鉴定特定的 BBB 测定，例如转铁蛋白受体转运蛋白活性、LPS 的 BBB 通透性挑战以及刺激诱导的白细胞迁移（目标 1）。成功标准是检测稳健性 Z' ≥ 0.2。第二阶段的目标2是开发人类BBB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 形成、渗透性和转运蛋白活性。为了证明实用性，该模型将使用 LPS、甘露醇和血管紧张素 II 进行处理，并评估 BBB 结构和功能的相关变化。这些化合物中的每一种都具有临床相关性，但通过不同的机制发挥作用。目标 3 是验证特定的人类 BBB 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显着促进基础、转化和临床神经科学研究的进展，并将显着推进对多种破坏性疾病的治疗。

 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.