Novel 3D high-content/throughput assay with mobile device-based data acquisition

描述（由申请方提供）：目前可用的毒性筛选模型并不总是准确预测人体毒性。动物模型是常用的，但它们是昂贵的，耗时的，道德上具有挑战性的，它们在物种之间变化，并且它们不能准确地预测人类的毒性。多年来，体外毒性测试一直被探索作为更便宜的替代品或作为体内测试之前的初始筛选，但仍然存在关于准确性的问题，主要是因为它们在二维（2D）表面上培养，而天然组织存在于三维环境（3D）中。因此，虽然伦理和成本动机驱使毒性筛选朝向体外模型，但目前在模拟天然组织中的体外测定的局限性阻止了它们的广泛接受和使用。该提案提出了一种快速、定量和具有代表性的高通量毒性测试3D模型。 最近，研究已经倾向于体外三维（3D）细胞培养系统，其在天然组织环境和反应中比2D系统更具代表性，并且比体内测试成本更低且更具争议性。3D的优势包括：（1）可溶性因子浓度的动态空间梯度;（2）更广泛的细胞-细胞和细胞-基质相互作用，以不同方式调节细胞功能和行为;（3）支持多种细胞类型的能力，具有空间组织以模拟天然环境。因此，用于毒性测试的3D细胞培养模型可以代表天然组织环境，并且比2D体外模型更好地预测体内毒性。然而，目前可用的3D细胞培养模型并不理想，因为这些模型昂贵，涉及大量的制造，并且分析起来耗时。例如，在一个可比较的模型中，3D球状体花费7-10天来测定细胞毒性。这些3D体外试验的长实验时间限制了研究的化合物数量，同时增加了与细胞培养相关的风险，如污染。 为此，该提案旨在设计一种基于3D人类细胞的体外试验，该试验能更好地代表感兴趣的人类组织，预测体内毒性，但在比其他试验更短的时间内完成。我们使用磁性纳米粒子，它是无毒的，可以用来使细胞具有磁性。这些磁化的细胞可以用磁力进行操纵，具有精细的空间控制，并且不需要任何特殊的设备或介质。在本提案中，我们将使用这种技术打印细胞 我们已经发现，这些环随着时间的推移而闭合/收缩，并且速率随化合物浓度而变化。这允许简单快速地打印3D细胞模型，用于毒性筛选。此外，我们将使用基于移动的设备的成像系统来对整个板进行成像，并且在这样做时，以显著较低的成本提高测定的效率和通量。在第一阶段，我们建议用3 T3小鼠胚胎成纤维细胞来验证我们的模型，根据NIEHS指南，3 T3小鼠胚胎成纤维细胞通常用于毒性测试，然后在第二阶段扩展到器官特异性毒性模型，特别是肺和肝脏。由此产生的3D毒性测定将利用3D细胞培养的优势，以快速、经济有效的方式更好地预测体外毒性。此外，该提案将开发基于移动的设备的分析工具，用于高通量分析。 我们的假设是，我们可以使用磁性打印设计一种新的体外3D测定，其将3D细胞培养的益处应用于毒性筛选，其比其他体外测定更好地预测体内毒性。这些测定将在不同条件下产生快速、定量、无标记的细胞迁移度量，以研究某些化合物的基础细胞毒性。该测定还将允许高通量分析以提高筛选通量和效率。此外，可以对3D培养物进行测定后实验，包括荧光染色，以研究特定的作用机制。在创建磁性打印3D检测时，我们将整合：快速打印3D细胞培养物的能力， 相关细胞外基质;实时和无标记定量环闭合，这与细胞功能相关;能够研究特定化合物的基础细胞毒性及其作用机制;用于高通量分析的工具，可以显着减少数据收集的时间和成本。该项目的最终结果是一种比其他检测方法更快，比动物模型和3D培养成本更低，比2D体外检测更具预测性的检测方法。这项提议得到了德克萨斯大学休斯顿健康科学中心、德克萨斯大学MD安德森癌症中心、莱斯大学、基因泰克和阿斯利康研究人员的支持信。本阶段SBIR提案的目标是：目标I：优化用于生物测定的3D细胞培养物的磁悬浮和图案化目标II：优化用于测量细胞毒性的生物测定目标III：优化生物测定中基于移动的设备的图像采集。

DESCRIPTION (provided by applicant): Currently available models for toxicity screening are not always accurate predictors of toxicity in humans. Animal models are commonly used, but they are costly, time-consuming, and ethically challenging, they vary between species, and they do not accurately predict toxicity in humans. In vitro toxicity tests have been explored for years as cheaper alternatives or as initial screens before in vivo testing, but there are still issues regarding accuracy, primarily because they are cultured on two-dimensional (2D) surfaces, while native tissues exist in three-dimensional environments (3D). As a result, while ethical and cost motivations drive toxicity screening towards in vitro models, the limitations of current in viro assays in mimicking native tissue have prevented their widespread acceptance and use. This proposal puts forward a 3D model that is rapid, quantitative, and representative for high-throughput toxicity testing. Recently, research has gravitated towards in vitro three-dimensional (3D) cell culture systems, which are more representative in native tissue environment and responses than 2D systems, and still less costly and controversial than in vivo tests. The advantages of 3D include: (1) dynamic spatial gradients of soluble factor concentrations; (2) a wider array of cell-cell and cell-matrix interactions that regulate cell function and behavior differently; and (3) the ability to support multiple cell types with spatial organization to mimic native environments. As a result, 3D cell culture models for toxicity testing could represent native tissue environments and predict in vivo toxicity better than 2D in vitro models. However, currently available 3D cell culture models are not ideal given that these models are expensive, involve extensive fabrication, and are time-consuming to analyze. For example, in one comparable model, 3D spheroids took 7-10 days to assay cytotoxicity. The long experimentation time of these 3D in vitro assays limits the number of compounds studied while increasing risks related to cell culture, like contamination. To that end, this proposal looks to design a 3D human cell-based in vitro assay that better represents the human tissue of interest, predicts in vivo toxicity, but does so within a shorter timeframe than other assays. We use magnetic nanoparticles, which are nontoxic, and can be used to render cells magnetic. These magnetized cells can then be manipulated with magnetic forces with fine spatial control, and without the need of any special equipment or media. In this proposal, we will use this technique to print cells into 3D rings, that we have found to close/contract over time, and at rates that vary with compound concentration. This allows for the easy and rapid printing of 3D cellular models for the purpose of toxicity screening. Additionally, we will use a mobile device-based imaging system to image whole plates, and in doing so, increasing efficiency and throughput of the assay at a significantly lower cost. In Phase I, we propose to validate our model with 3T3 mouse embryonic fibroblasts, which are commonly used for toxicity testing, according to NIEHS guidelines, before expanding into organ-specific toxicity models, specifically of the lung and liver, in Phase II. The resulting 3D toxicity assay from this proposal will use the advantages of 3D cell culture to better predict in vitro toxicity in a quick, cost-effective fashion. In addition this proposal will develop mobile- device based analytical tools for high-throughput analysis. Our hypothesis is that we can design a novel in vitro 3D assay using magnetic printing that applies the benefits of 3D cell culture towards toxicity screening that better predicts in vivo toxicity thn other in vitro assays. These assays would yield fast, quantitative, label-free metrics of cell migration under different conditions to study the basal cytotoxicity of certain compounds. This assay would also allow for high-throughput analysis to improve screening throughput and efficiency. In addition, post-assay experimentation, including fluorescent staining, can be performed on the 3D cultures to investigate particular mechanisms of action. In creating a magnetically printed 3D assay, we will integrate: Capability to rapidly print 3D cell cultures with relevant extracellular matrix; Real-time and label-free quantification of ring closure, which correlates with cell function; Ability to investigate the basal cytotoxicity of particular compound and their mechanisms of actions; Tools for high-throughput analysis that could significantly cut the time and cost of data collection. The end result of this project is an assay that is faster tha other assays, less costly than animal models and 3D cultures, and more predictive than 2D in vitro assays. This proposal has letters of support from researchers at University of Texas Health Science Center - Houston, University of Texas MD Anderson Cancer Center, Rice University, Genentech, and AstraZeneca. The aims of this Phase I SBIR proposal are: Aim I: Optimize the magnetic levitation and patterning of 3D cell cultures for the BiO Assay Aim II: Validate the BiO Assay for measuring cytotoxicity Aim III: Validate the mobile device-based image acquisition in the BiO Assay.