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

项目总结： 供人类使用或潜在的人类接触的新化合物的神经毒理学评估 由国际监管机构授权，主要依赖于对高级脊椎动物的致命性测试 动物。较高的筛选成本、较长的实验时间和减少对 动物试验导致许多行业寻找替代技术。体外毒理学检测的用途 分离细胞或单型细胞培养，只能提供有限的洞察力，因为这些模型缺乏生物学意义 相关完整的多类型蜂窝网络结构。虽然这两种技术都得到了硅胶技术的增强 技术方面，从简单、快速、 与更长、复杂和昂贵的高阶动物体内研究相比，体外方法成本更低。 Newormics填补这一空白的方法是实现对线虫病的体内神经毒理学评估 秀丽，一种公认的替代无脊椎动物模式生物，通过开发神经元特异性毒性测试， 通过专有的高密度、大规模微流控固定化设备提供，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 三个具体目标中的目标。在目标1中，我们将把我们的第一代微流控设备转换为高密度(384- 好的)带有改进的微制造技术的活芯片，结合了芯片上的培养，实现了免转移 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 显著增加测试规模(从每个芯片80个化合物增加到280个)，并降低消耗品和人力成本 每一次测试。在目标2中，在我们第一阶段的多巴胺能神经毒素分析的基础上，我们将开发四种神经毒素 多巴胺能、5-羟色胺能、GABA能和胆碱能神经元的荧光分析 提供前所未有的能力来评估化学品对完好无损个体的微妙表型影响， 功能神经元。以实现实时图像处理、多参数表型和管理 每个测试生成TB级的图像数据，我们将构建一个由图形用户支持的计算平台 界面。该平台将用于图像编辑、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AIM 3，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检测结果。随着更多的化学品被测试，我们将建立一个 可以进一步挖掘的数据库。这项工作的结果将使许多行业能够减少致命的 动物试验和让更安全的工业和个人消费产品更快地投放市场，以获得经济效益， 达到减少动物使用的监管合规性，并改善神经疾病的医疗保健。

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.