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.

项目概要： 对供人类使用或人类潜在接触的新化合物进行神经毒理学评估 由国际监管机构强制执行，很大程度上依赖于高阶脊椎动物的致死率测试 动物。筛选成本高、实验时间长以及减少依赖的立法要求 动物试验促使许多行业寻找替代技术。体外毒理学测试用途 分离的细胞或单型细胞培养物，只能提供有限的见解，因为这些模型缺乏生物学 相关完整的多类型细胞网络结构。虽然这两种技术都通过计算机增强 就技术而言，从简单、快速、 廉价的体外方法与在高级动物中进行的更长、复杂且昂贵的体内研究相比。 Newormics 填补这一空白的方法是对秀丽隐杆线虫进行体内神经毒理学评估 线虫是一种公认​​的替代无脊椎动物模型生物，通过开发神经元特异性毒性测定， 通过专有的高密度、大规模微流体固定装置提供，用于高含量、高 吞吐量分析。以第一阶段取得的进展和重要的市场经验为基础 参与 NIH I-Corps 计划，第二阶段提出了几个新的创新要素，以实现我们的目标 3个具体目标的目标。在目标 1 中，我们将把第一代微流体装置转换为高密度（384- 好）vivoChip 采用改进的微加工技术，整合了片上培养物以实现无转移 曝光和测试，并集成芯片加载、成像和分析的自动化。这些措施将 显着增加测试规模（从每芯片 80 种化合物增加到 280 种）并降低耗材和劳动力成本 每次测试。在目标 2 中，在第一阶段的多巴胺能神经毒素测定的基础上，我们将开发四种神经毒素 使用明亮荧光标记的多巴胺能、血清素能、GABA 能和胆碱能神经元进行测定 提供前所未有的能力来评估化学物质对个体完整的微妙表型影响， 功能神经元。实现实时图像处理、多参数表型分析和管理 每次测试生成 TB 的图像数据，我们将构建一个由图形用户授权的计算平台 界面。该平台将用于图像编译、用户注释的表型定义和评分以及 通过适当的统计分析自动生成报告。在目标 3 中，我们将与我们的行业合作伙伴一起 使用参考化学品验证我们的平台和检测。随着更多化学品的测试，我们将建立一个 可以进一步挖掘的数据库。这项工作的成果将使许多行业能够减少致命的 动物测试并让更安全的工业和个人消费品更快地上市以获得经济效益， 达到减少动物使用的监管合规性，并改善神经系统疾病的医疗保健。