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

项目摘要： 拟用于人类或潜在人类暴露的新化合物的神经毒理学评价是 由国际监管机构强制执行，主要依赖于高阶脊椎动物的致死率测试 动物高筛选成本，长实验时间，以及立法要求，以减少依赖 动物试验已使许多工业寻找替代技术。体外毒理学试验用途 分离的细胞或单型细胞培养物，并且只能提供有限的洞察力，因为这些模型缺乏生物学特性， 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技术得到了增强， 技术，仍然有一个不平凡的差距，可以学到什么，从简单，快速， 廉价的体外方法与较长、复杂和昂贵的高等动物体内研究相比。 Newormics填补这一空白的方法是在小杆线虫中进行体内神经毒理学评估 elegans，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定， 通过专有的高密度、大规模微流体固定化装置输送， 吞吐量分析在第一阶段取得的进展和从 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。 三个具体目标。在目标1中，我们将把我们的第一代微流控装置转换为高密度（384- 以及）vivoChip与改进的微加工技术，结合芯片上的培养， 曝光和测试，以及芯片装载、成像和分析的集成自动化。这些措施将 显著增加测试规模（从每个芯片80种化合物增加到280种），降低耗材和劳动力成本 每次测试。在目标2中，基于我们第一阶段的多巴胺能神经毒性试验，我们将开发四种神经毒性药物， 使用明亮荧光标记的多巴胺能、多巴胺能、GABA能和胆碱能神经元的测定 提供了前所未有的能力来评估化学品对个体完整的微妙表型影响， 功能性神经元为了实现实时图像处理、多参数表型分型和管理 我们将建立一个由图形用户授权的计算平台， 接口.该平台将用于图像编辑、用户注释的表型定义和评分，以及 自动生成报告，并进行适当的统计分析。在目标3中，我们将与行业合作伙伴一起， 使用参考化学品验证我们的平台和分析。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。这项工作的成果将使许多行业能够减少致命的 动物试验和获得更安全的工业和个人消费产品更快地推向市场，以获得经济效益， 达到减少动物使用的法规合规性，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.