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

描述(由申请人提供)： 这项研究的总体目标是开发一种新型的体外微流控平台，以测试药物或给药载体渗透血脑屏障(BBB)的能力。与目前的体外模型不同，我们提出的SIM-BBB装置由一个微流控两室室组成。该试验室的设计允许在适当的微循环大小和流动条件下对传输/渗透进行可视化友好的评估，同时简化设备制造。顶侧种植内皮细胞，基底外侧支持神经胶质细胞共培养。生理现实主义的增强大大改善了血脑屏障的特征，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、更大的图书馆覆盖率、更低的成本、更快的周转时间和更多有利于药物发现工作的机械知识。在第一阶段，设计并制造了第一代微流控SIM-BBB器件。在含有星形胶质细胞条件培养液的微流控结构中培养脑内皮细胞。生化分析显示紧密连接分子上调，而光学分析显示微流控装置中的血脑屏障完整。最后，在该装置上成功地实现了转运蛋白的检测。第二阶段的工作将集中于优化微流控设备，以增强生理保真度。将整合电极，通过跨内皮细胞电阻(TEER)测量，对内皮细胞层和紧密连接的形成进行非目视监测。最后，开发的技术将被用于不同的应用，包括药物渗透研究和炎症条件下的白细胞迁移。为顺利完成该项目，已经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。

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.