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

通过基于移动设备的数据采集进行新型 3D 高内涵/通量测定

• 批准号: 8781697
• 负责人: Glauco Ranna Souza
• 金额: $ 15万
• 依托单位: NANO3D BIOSCIENCES, INC.
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-01 至 2016-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8781697
• 关键词: 3D Print; Acetaminophen; Albumins; Animal Model; Bile Acids; Biological Assay; Cell Culture System; Cell Culture Techniques; Cell Proliferation; Cell Survival; Cell model; Cell physiology; Cells; Cellular Structures; Color; Contracts; Cytochrome P450; Cytoskeletal Modeling; Data; Data Analyses; Data Collection; Dose; Doxorubicin; Drops; Embryo; Environment; Ethics; Extracellular Matrix; F-Actin; Fibroblasts; Gene Expression; Goals; Guidelines; Health Sciences; Hepatotoxicity; Hour; Human; Ibuprofen; Image; In Vitro; Inhibitory Concentration 50; Label; Laboratory Chemicals; Letters; Life; Liver; Lung; Magnetism; Measures; Methods; Metric; Microscope; Modeling; Motivation; Mucins; Mus; National Institute of Environmental Health Sciences; Organ; Paclitaxel; Pattern; Pharmaceutical Preparations; Pharmacologic Substance; Phase; Printing; Production; Reporting; Reproducibility; Research; Research Personnel; Resolution; Rice; Risk; Science; Side; Small Business Innovation Research Grant; Special Equipment; Staining method; Stains; Surface; System; Techniques; Testing; Texas; Time; Tissues; Toxic effect; Toxicity Tests; Tubulin; Universities; University of Texas M D Anderson Cancer Center; Vimentin; alpha-Fetoproteins; analytical tool; base; cell behavior; cell motility; cell type; cost; cost effective; cytotoxic; cytotoxicity; data acquisition; design; digital; handheld mobile device; high throughput analysis; human tissue; improved; in vitro Assay; in vitro Model; in vitro testing; in vivo; interest; kidney cell; meetings; migration; mimicry; nanoparticle; novel; prevent; public health relevance; research study; response; screening; success; three-dimensional modeling; tool; two-dimensional

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.

描述(由申请人提供)：目前可用于毒性筛选的模型并不总是对人体毒性的准确预测。动物模型是常用的，但它们昂贵、耗时和具有伦理挑战性，不同物种的动物模型不同，而且它们不能准确预测对人类的毒性。多年来，体外毒性测试一直被探索为更便宜的替代品或作为体内测试之前的初始筛选，但仍存在准确性问题，主要是因为它们是在二维(2D)表面培养的，而天然组织存在于三维环境(3D)中。因此，尽管伦理和成本动机促使毒性筛选转向体外模型，但目前病毒检测在模拟天然组织方面的局限性阻碍了它们的广泛接受和使用。该方案提出了一种快速、定量、具有代表性的高通量毒性试验三维模型。近年来，体外三维(3D)细胞培养系统成为研究的热点，这种系统在自然组织环境和反应中比2D系统更具代表性，而且比体内试验更便宜，也更具争议性。3D的优势包括：(1)可溶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，以不同的方式调节细胞的功能和行为；(3)能够支持多种细胞类型，其空间组织模拟自然环境。因此，用于毒性测试的3D细胞培养模型可以代表自然组织环境，并比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，涉及广泛的制造，并且分析起来很耗时。例如，在一个可比的模型中，3D球体需要7-10天来检测细胞毒性。这些3D体外分析的长时间实验限制了所研究的化合物的数量，同时增加了与细胞培养相关的风险，如污染。为此，该提案希望设计一种基于3D人体细胞的体外测试，更好地代表感兴趣的人体组织，预测体内毒性，但比其他测试在更短的时间内做到这一点。我们使用磁性纳米颗粒，这是无毒的，可以用来使细胞具有磁性。然后，这些磁化的电池可以通过磁力进行精细的空间控制，而不需要任何特殊的设备或介质。在本提案中，我们将使用此技术打印单元格 进入3D环，我们发现随着时间的推移，这些环会关闭/收缩，并且速度会随着化合物浓度的变化而变化。这使得为了毒性筛选的目的，可以轻松快速地打印3D细胞模型。此外，我们将使用基于移动设备的成像系统来对整个平板进行成像，这样做将以显著较低的成本提高分析的效率和吞吐量。在第一阶段，我们建议用3T3小鼠胚胎成纤维细胞来验证我们的模型，根据NIEHS指南，3T3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。根据这一建议产生的3D毒性测试将利用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，该提案还将开发基于移动设备的分析工具，用于高通量分析。我们的假设是，我们可以设计一种使用磁性打印的新型体外3D检测方法，将3D细胞培养的优点应用于毒性筛选，比其他体外检测方法更好地预测体内毒性。这些分析方法将产生快速、定量、无标记的不同条件下的细胞迁移指标，以研究某些化合物的基础细胞毒性。这项检测还将允许高通量分析，以提高筛选吞吐量和效率。此外，可以在3D培养物上进行包括荧光染色在内的检测后实验，以研究特定的作用机制。在创建磁性打印3D分析时，我们将集成：快速打印3D细胞培养的能力与 相关的细胞外基质；与细胞功能相关的环关闭的实时和无标记量化；调查特定化合物的基础细胞毒性及其作用机制的能力；可大幅削减数据收集时间和成本的高通量分析工具。该项目的最终结果是一种比其他测试更快、比动物模型和3D培养成本更低、比2D体外测试更具预测性的测试。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的研究人员的支持信。第一阶段SBIR提案的目的是：目标一：优化生物检测中3D细胞培养的磁悬浮和构图。目标二：验证生物检测中用于测量细胞毒性的生物检测方法。目标三：在生物检测中验证基于移动设备的图像采集。

项目成果

A spheroid toxicity assay using magnetic 3D bioprinting and real-time mobile device-based imaging.

• DOI: 10.1038/srep13987
• 发表时间: 2015-09-14
• 期刊: Scientific reports
• 影响因子: 4.6
• 作者: Tseng H;Gage JA;Shen T;Haisler WL;Neeley SK;Shiao S;Chen J;Desai PK;Liao A;Hebel C;Raphael RM;Becker JL;Souza GR
• 通讯作者: Souza GR

Assembly of Hepatocyte Spheroids Using Magnetic 3D Cell Culture for CYP450 Inhibition/Induction.

• DOI: 10.3390/ijms18051085
• 发表时间: 2017-05-18
• 期刊: International journal of molecular sciences
• 影响因子: 5.6
• 作者: Desai PK;Tseng H;Souza GR
• 通讯作者: Souza GR