A novel physiologically realistic microfluidic in-vitro blood-brain barrier model

描述（由申请人提供）： 本研究的总体目标是开发一种新型的体外微流控平台，以测试药物或递送载体渗透血脑屏障（BBB）的能力。与目前的体外模型相比，我们提出的装置，SIM-BBB，包括一个微流体双室。该室的设计方式是允许在适当的微循环尺寸和流动条件下对运输/渗透进行可视化友好的评估，同时简化设备制造。顶侧接种内皮细胞，基底侧支持神经胶质细胞共培养。增加的生理现实性显著改善了BBB特征，包括紧密连接的形成和相关转运蛋白的表达。新平台提供了更大的吞吐量，增加了库覆盖率，降低了成本，快速周转时间和增加了有利于药物发现工作的机制知识。在第一阶段，第一代微流控SIM-BBB设备的设计和制造使用软光刻。脑内皮细胞在具有星形胶质细胞条件培养基的灌注液的微流体构建体中培养。生化分析显示紧密连接分子的上调，而光学分析显示微流控装置中BBB的完整性。最后，转运蛋白测定在该装置中被成功地证实。第二阶段的工作将集中在优化微流体装置，以提高生理保真度。将整合电极，通过跨内皮电阻（TEER）测量对内皮细胞层和紧密连接形成进行非目视监测。最后，开发的技术将被证明用于不同的应用，包括药物渗透研究和炎症条件下的白细胞迁移。为成功完成该项目，已组建了一个具有工程和生物学专业知识的多学科伙伴关系。 公共卫生相关性： 该项目旨在开发一种体外筛选模型，用于筛选候选药物穿过血脑屏障并随后引起治疗或毒性作用的潜力。通过提供准确和预测性的数据，该模型将减少对动物模型的需求，并有望减少晚期候选药物的失败，加速中枢神经系统（CNS）治疗的开发。该产品将商业化给制药公司，药物研究实验室和大学/非营利中心从事新的神经治疗研究和中枢神经系统毒性。同样重要的是，它也有望刺激基础研究，在那里它可以被用来研究BBB（dys）功能的生物学机制。

DESCRIPTION (provided by applicant): The overall objective of this study is to develop a novel in vitro microfluidic platform to test a drug or delivery vehicle's ability to permeate the Blood-Brain Barrier (BBB). In contrast to current in-vitro models, our proposed device, SIM-BBB, comprises of a microfluidic two-compartment chamber. The chamber is designed in such a way as to permit visualization-friendly evaluation of transport/permeation under appropriate microcirculatory size and flow conditions, while simultaneously simplifying device fabrication. The apical side is seeded with endothelial cells and the basolateral side supports glial cell co-cultures. The increased physiological realism substantially improves BBB characteristics including formation of tight junctions and expression of relevant transporters. The new platform offers greater throughput, increased library coverage, lower cost, rapid turnaround times and increased mechanistic knowledge benefiting drug discovery efforts. In Phase I, the first generation microfluidic SIM-BBB device was designed and fabricated using soft lithography. Brain endothelial cells were cultured in the microfluidic constructs with a perfusate of astrocyte conditioned media. Biochemical analysis showed upregulation of tight junction molecules while optical analysis showed intactness of the BBB in the microfluidic device. Finally, transporters assay was successfully demonstrated in the device. Phase II efforts will focus on optimization of the microfluidic device for enhanced physiological fidelity. Electrodes will be integrated for non-visual monitoring of the endothelial cell layers and tight junction formation via trans-endothelial electrical resistance (TEER) measurements. Finally, the developed technology will be demonstrated for diverse applications including drug penetration studies and leukocyte migration under inflammatory conditions. A multi-disciplinary partnership with expertise in engineering and biology has been assembled for successful completion of the project. PUBLIC HEALTH RELEVANCE: The project seeks to develop an in vitro screening model for screening the potential of drug candidates to cross the BBB and subsequently cause therapeutic or toxic effects. By providing accurate and predictive data, the model will reduce the need for animal models and promises to both reduce late stage drug candidate failures and accelerate central nervous system (CNS) therapeutic development. The product will be commercialized to pharmaceutical firms, drug research labs and universities/non-profit centers engaged in novel neurological therapeutics research and CNS toxicity. Equally important, it is also expected to spur basic research, where it can be used to study the biological mechanisms of BBB (dys) function.