Myelinated Rodent Brain Spheroids for High-throughput Evaluation of Neurotoxicity

中枢神经系统（CNS）毒性可导致动物多种功能和行为改变 模型，如认知障碍、学习/记忆丧失、听力/视力丧失、感觉/运动功能改变 反射和其他一些功能障碍。虽然行为测试和动物模型足够敏感，可以预测 人类神经毒性，动物试验的费用和劳动密集型使得筛选大规模 化合物的数量。虽然在体外环境中进行行为测试是不可能的，但一些 可以评估的参数包括神经元和神经胶质细胞体的细胞毒性、脱髓鞘或改变的神经细胞毒性。 神经元放电模式，已知许多毒素会影响并最终导致行为/认知 赤字诱导多能干细胞技术的最新进展导致了人类胚胎干细胞的发展。 基于细胞的微生理学系统，作为先进的细胞模型， 可以提供高通量和高内容的数据，用于毒素筛选。虽然人类类器官 预计预测人类神经毒性比动物模型更好，使用人类类器官生成的数据 可能不够可靠，除非研究表明体外神经毒性成功预测体内皮质 历史动物研究中显示的功能障碍。为了克服这些挑战， 通过人脑器官型培养系统获得，我们开发了一种创新的功能成熟的3D 通过培养胚胎分离的啮齿动物脑球状体模型的一致大小和不变的组成 皮层组织 第一阶段研究的目的是证明某些化学毒素 啮齿类动物的神经毒性将在微工程神经组织中诱导毒性， 图像分析、神经元放电模式和组织形态计量学。这一目标将通过量化离散 生物学和电学可量化的指标，通过这些指标可以证明未经治疗的脑球状体与 与对照组相比，差异显著。 这一目标将分两个阶段实现：研究计划将包括第一次开发和 使用高通量共聚焦筛选和微电极阵列表征3D啮齿动物脑球状体 用于基线生物学和功能评估。(Aim 1）。然后，我们将比较生物学和 培养的未经处理的球状体与环境激素处理的球状体的电生理特性 毒素，以验证在存在损伤的情况下的测定（目标2）。 这些目标的成功完成将有力地将该技术定位为第二阶段奖，其中 我们将寻求扩大制造和测试规模，并充分验证临床- 与动物数据相比，具有更大的化合物库的类似度量。我们将进一步执行 “组学”研究，并将最终使用这种测定方法来确定大范围的毒理学参数 从而理解作用机制以及改善对生物过程的理解。

Central nervous system (CNS) toxicity results in a wide variety of functional and behavioral changes in animal models such as cognitive impairments, learning/memory loss, hearing/vision loss, alterations in sensory/motor reflex, and several other dysfunctions. While behavioral tests and animal models are sensitive enough to predict human neurotoxicity, the expense and labor-intensiveness of animal testing make it challenging to screen large numbers of chemical compounds. While running behavioral tests is not possible in in vitro setting, some parameters that can be evaluated include cytotoxicity of neuronal and glial cell bodies, demyelination, or altered neuronal firing patterns, which many toxins are known to impact and ultimately results in behavioral/cognitive deficits. Recent advances in induced pluripotent stem cell technology have resulted in the development of human cells-based microphysiological systems that have shown tremendous promise as advanced cellular models that can provide high-throughput and high-content data useful for toxin screening. While human organoids are expected to predict human neurotoxicity better than animal models, the data generated using human organoids may not be reliable enough unless studies show that in vitro neurotoxicity successfully predicts in vivo cortical dysfunctions shown in the historical animal studies. To overcome these challenges and strengthen findings obtained by human brain organotypic culture systems, we have developed an innovative functionally mature 3D rodent brain spheroid model of consistent size and unvarying composition by culturing embryonic dissociated cortical tissue. The objective of this phase I study is to demonstrate that certain chemical toxins known for causing neurotoxicity in rodents will induce toxicity in micro-engineered neural tissue that can be quantified using 3D image analysis, neuronal firing patterns and histomorphometry. This goal will be achieved by quantifying discrete biological and electrical quantifiable metrics by which untreated brain spheroids can be demonstrated to differ significantly compared to treated controls. This goal will be accomplished in two phases: The research plan will consist of first developing and characterizing 3D rodent brain spheroids using high throughput confocal screening and microelectrode arrays for baseline biological and functional evaluation. (Aim 1). Then, we will compare biological and electrophysiological characteristics of cultured untreated spheroids with spheroids treated with environmental toxins to validate the assay in the presence of an insult (Aim 2). Successful completion of these aims will strongly position this technology for a Phase II award, in which we will seek to scale up fabrication and testing and fully validate the specificity and sensitivity of the clinically- analogous metrics with a larger library of compounds, as compared with animal data. We will further perform “omics” studies and will eventually use this assay for determining a large spectrum of toxicological parameters resulting in understanding mechanisms of action as well as improved understanding of biological processes.