A Multiwell Plate Format Microfluidic Immobilization Chip for High-Content Imaging of Whole Animals for in vivoNeurotoxicology Testing

多孔板形式微流体固定芯片，用于对整个动物进行体内神经毒理学测试的高内涵成像

• 批准号: 10428522
• 负责人: Evan Hegarty
• 金额: $ 74.99万
• 依托单位: VIVOVERSE, LLC
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-04-01 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10428522
• 关键词: 3-Dimensional; Address; Animal Model; Animal Testing; Animals; Automation; Biological; Biological Assay; Caenorhabditis elegans; Cell Culture Techniques; Cells; Chemicals; Complement; Complex; Computer software; Data; Databases; Dendrites; Dependence; Development; Devices; Dopamine; Eating; Economics; Elements; Evaluation; Exposure to; Feedback; Future; Gel; Generations; Goals; Healthcare; Hour; Human; Image; Immobilization; In Vitro; Individual; Industrialization; Industry; Industry Standard; Innovation Corps; International; Invertebrates; Label; Learning; Liquid substance; Machine Learning; Manuals; Market Research; Measures; Methods; Microfabrication; Microfluidic Microchips; Microfluidics; Modeling; Nerve Degeneration; Nervous system structure; Neurons; Neurotransmitters; Organoids; Outcome; Phase; Phenotype; Population; Positioning Attribute; Process; Protocol Compliance; Protocols documentation; Reporting; Research Activity; Resolution; Robotics; Serotonin; Services; Specificity; Speed; Statistical Data Interpretation; Structure; System; Technology; Testing; Time; Toxic effect; Toxicity Tests; Toxicology; Translating; United States National Institutes of Health; Validation; Variant; Vertebrates; Whole Organism; Work; base; cholinergic neuron; computational platform; connectome; consumer product; cost; cost effective; density; design; developmental neurotoxicity; developmental toxicity; empowered; experimental study; exposed human population; fluorescence imaging; gamma-Aminobutyric Acid; graphical user interface; high resolution imaging; high throughput analysis; image processing; imaging platform; imaging system; improved; in silico; in vitro Model; in vivo; in vivo Model; industry partner; innovation; insight; interest; nervous system disorder; neurotoxicity; neurotoxicology; programs; real-time images; reproductive toxicity; screening; small molecule libraries; success; terabyte; testing services; young adult

Project Summary: Neurotoxicological evaluation of new compounds intended for human use or of potential human exposure is mandated by international regulatory bodies and largely relies on lethality testing in higher-order vertebrate animals. High screening costs, long experimental times, and legislative requirements to reduce dependence on animal testing have led many industries to search for alternative technologies. In vitro toxicology testing uses isolated cells or monotypic cell culture and can only provide limited insight since these models lack biologically relevant intact multi-typic cellular network structures. While both technologies have been augmented by in silico technologies, there is still a non-trivial gap between what can be learned and translated from simple, fast, inexpensive in vitro methods versus longer, complex, and costly in vivo studies in higher order animals. Newormics’ approach to filling this gap is to enable in vivo neurotoxicological assessment in Caenorhabditis elegans, an accepted alternative invertebrate model organism, by developing neuron-specific toxicity assays, delivered via a proprietary high-density, large-scale microfluidic immobilization device for high-content, high throughput analysis. Building on advances made during Phase I and important market learnings from participation in the NIH I-Corps program, Phase II proposes several new elements of innovation to achieve our goals in 3 specific aims. In Aim 1, we will convert our first-generation microfluidic device to a high-density (384- well) vivoChip with improved microfabrication technologies, incorporate on-chip culture for transfer-less exposure and testing, and integrate automation for chip loading, imaging, and analysis. These measures will significantly increase test scale (from 80 compounds per chip to 280) and lower the consumable and labor costs per test. In Aim 2, building on our dopaminergic neurotox assay from Phase I, we will develop four neurotox assays with brightly fluorescently labeled dopaminergic, serotonergic, GABAergic, and cholinergic neurons providing the unprecedented ability to assess subtle phenotypic effects of chemicals on individual intact, functional neurons. To achieve real-time image processing, multi-parameter phenotyping, and managing the terabytes of image data generated per test, we will build a computational platform empowered by a graphic user interface. This platform will be used for image compilation, user-annotated phenotype definition and scoring, and automated report generation with appropriate statistical analysis. In Aim 3, with our industry partners, we will validate our platform and assays using reference chemicals. As more chemicals are tested, we will build a database which can be further mined. The outcome of this work will enable many industries to reduce lethal animal testing and get safer industrial and personal consumer products to market faster for economic benefit, reaching regulatory compliance for reduced animal use, and improved healthcare for neurological diseases.

项目摘要： 针对人类使用或潜在人类暴露的新化合物的神经毒理学评估是 国际监管机构的要求，很大程度上依赖于高阶脊椎动物的致死性测试 动物。较高的筛查成本，漫长的实验时间和立法要求，以减少对 动物测试已导致许多行业寻找替代技术。体外毒理学测试用途 孤立的细胞或单型细胞培养，只能提供有限的见解，因为这些模型在生物学上缺乏 相关完整的多型蜂窝网络结构。虽然这两种技术都在计算机中得到了增强 技术，可以从简单，快速，快速，快速地学习的内容之间存在非平凡的差距 廉价的体外方法与更长，复杂且昂贵的体内研究中的体内研究。 填补这一差距的新学方法是使caenorhabditis的体内神经毒理学评估能够实现体内神经毒理学评估 秀丽隐杆线虫是一种通过开发神经元特异性毒性测定的公认替代无脊椎动物模型生物体， 通过专有的高密度，大规模的微流体固定装置提供，用于高容量，高 吞吐量分析。以第一阶段的进步和重要的市场学习为基础 参与NIH I-Corps计划，第二阶段提案为创新的几个新元素以实现我们 三个特定目标的目标。在AIM 1中，我们将将第一代微流体设备转换为高密度（384-- 井）vivochip具有改进的微加工技术，并结合了片上培养物，用于转移 暴露和测试，以及用于芯片加载，成像和分析的集成自动化。这些措施将 显着提高测试量表（从每芯片80种化合物增加到280种），并降低了易消耗品和人工成本 按测试。在AIM 2中，基于第一阶段的多巴胺能神经毒素测定法，我们将开发四个神经毒素 具有明亮荧光标记的多巴胺能，血清素能，GABA能和胆碱能神经元的测定 提供了评估化学物质对个体完整的微妙表型影响的前所未有的能力， 功能性神经元。实现实时图像处理，多参数表型和管理 每次测试生成的图像数据的Terabytes，我们将构建一个由图形用户授权的计算平台 界面。该平台将用于图像汇编，用户声明的表型定义和评分，以及 自动报告生成，并进行适当的统计分析。在AIM 3中，与我们的行业合作伙伴一起，我们将 使用参考化学品验证我们的平台和测定。随着越来越多的化学物质的测试，我们将建造一个 可以进一步开采的数据库。这项工作的结果将使许多行业能够减少致命 动物测试并获得更安全的工业和个人消费产品，以更快地销售经济利益， 达到减少动物使用的法规依从性，并改善了神经系统疾病的医疗保健。