A High-Throughput Microfluidic in vitro CNS Myelination Model towards Drug Screening

 描述(由申请人提供)：中枢神经系统(CNS)的正常功能严重依赖于轴突周围绝缘髓鞘的形成。髓鞘形成是一个复杂的多步骤过程，主要发生在出生后早期，但也可以在成人中枢神经系统重新启动，以响应急性脱髓鞘侮辱。髓鞘形成细胞功能障碍和/或髓鞘丧失是许多神经系统疾病的基础，包括多发性硬化症(MS)和精神分裂症等心理障碍。促进髓鞘修复和实现神经保护已引起越来越多的关注，包括我们在内的几家制药公司和研究实验室正在积极寻找促进这一过程的类药物小分子。然而，这一努力的主要障碍之一是缺乏强大的体外中枢神经系统髓鞘形成模型。我们的多学科研究团队最近开发了一种微流控CNS神经干细胞/祖细胞(NSPC)聚合培养系统，该系统首次使用微型设备在体外展示了皮质神经元的强大髓鞘包裹。这种聚合培养系统的主要优点是它包含了体内环境的所有必要的细胞成分。然而，这种微型设备的使用是劳动密集型的，并且吞吐量相当有限。在这里，我们建议开发一种高通量微流控NSPC聚合培养系统，该系统提供的吞吐量至少比包括我们在内的现有方法高两个数量级，首次建立了具有生理相关神经元反应并适用于药物筛选应用的高通量体外中枢神经系统髓鞘形成模型。创新的三维高通量筛选(3D-HTS)微系统将至少有三套100个独立控制的微型培养舱(总共300个)，每个舱有8个聚集诱捕部位。全自动系统将能够筛选20个候选药物分子的效果，每个分子有5个不同的浓度，每个条件有3个重复，所有这些都是在一次实验运行中完成的。这个 微流控系统的配置很灵活，可以很容易地重新配置，以筛选大量的候选药物分子，而测试的浓度较少。由于图像处理是高通量筛选的另一个主要瓶颈，我们将开发一种图像处理算法，将髓鞘片段量化所需的图像数量降至最低，并完全自动化将生成的大量免疫染色图像的图像处理步骤。我们将使用这个微流控系统进行概念验证药物筛选试验。这将是第一个体外髓鞘系统 概述了皮质轴突-神经胶质的相互作用，并完全适应于整个药物筛选分析的自动化。我们预计，这一高通量平台的成功开发将导致建立一个常规的药物筛选系统，以识别潜在的靶点或候选药物分子，这些药物分子可以刺激髓鞘修复，帮助恢复脱髓鞘疾病和其他神经疾病的功能。

 DESCRIPTION (provided by applicant): Proper functioning of the central nervous system (CNS) is critically dependent on the formation of insulating myelin sheaths around axons. Myelination is a complex multi-step process that occurs primarily during early postnatal life, but it is also re-initiated in the adult CNS in response to acute demyelination insults. Dysfunction o myelin-forming cells and/or loss of myelin sheath underlie many neurological disorders including multiple sclerosis (MS) and psychological disorders such as schizophrenia. Promoting myelin repair and achieving neuroprotection has attracted increasing attention, and several pharmaceutical companies and research laboratories including ours are actively pursuing the discovery of drug-like small molecules that promote such processes. However, one of the major roadblocks in this effort is the lack of robust in vitro CNS myelination models. Our multidisciplinary research team has recently developed a microfluidic CNS neural stem/progenitor cell (NSPC) aggregate culture system that for the first time demonstrated robust myelin ensheathment of cortical neurons in vitro using a microdevice. The major advantages of this aggregate culture system are that it contains all the essential cellular components of an in vivo environment. However the use of this microdevice is labor intensive and throughput is quite limited. Here we propose to develop a high-throughput microfluidic NSPC aggregate culture system that provides at least two orders of magnitude higher throughput than existing approaches including ours, establishing for the first time a high-throughput in vitro CNS myelination model that has physiologically relevant neuronal responses and is amenable to drug screening applications. The innovative three-dimensional high-throughput screening (3D-HTS) microsystem will have at least three sets of 100 independently controlled microscale culture compartments (300 total), each compartment having eight aggregate trapping sites. The fully automated system will enable screening the effects of 20 candidate drug molecules at 5 different concentrations each, with 3 repeats per condition, all in a single experimental run. The microfluidic system configuration is flexible, where it can be easily re-configured to screen large number of candidate drug molecules with fewer number of concentrations tested. As image processing is another major bottleneck for high-throughput screening, we will develop an image processing algorithm that will minimize the number of images required for myelin segment quantification and fully automate the image processing steps for the large number of immunostained images that will be generated. We will conduct a proof-of-concept drug screening assay using this microfluidic system. This will be the first in vitro myelinating system recapitulating cortical axon-glial interactions and one that is fully adaptable to automation of th entire drug screening assay. We expect that the successful development of this high-throughput platform will lead to a routine drug screening system for identifying potential targets or candidate drug molecules that can stimulate myelin repair and help recover function in demyelinating diseases and other neurological disorders.