A novel 3D cell culture human uterine contractility assay for high-throughput scr

一种新型 3D 细胞培养人子宫收缩力测定，用于高通量 SCR

• 批准号: 8781654
• 负责人: Biana Godin
• 金额: $ 19.05万
• 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: 2014-09-22 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8781654
• 关键词: 3D Print; Affect; Animal Model; Animals; Area; Atosiban; Bathing; Biological; Biological Assay; Birth; Blood Circulation; Cell Count; Cell Culture Techniques; Cell Line; Cell Survival; Cell model; Cell physiology; Cells; Clinical Data; Collagen; Computer software; Contracts; Controlled Environment; Cytochrome P450; Cytoskeleton; Data; Data Collection; Development; Disease; Dose; Drug Kinetics; Dyes; Elastin; Elements; Environment; Equipment; Ethical Issues; Exposure to; Extracellular Matrix; F-Actin; Fibroblasts; Gene Expression; Glass; Goals; Hour; Human; Human Activities; Ibuprofen; Image; In Vitro; Incubated; Individual; Indomethacin; Label; Labor Onset; Lead; Life; Magnetism; Measures; Methodology; Methods; Metric; Microscope; Modeling; Molecular Conformation; Monitor; Mus; Muscle Contraction; Myometrial; Myosin ATPase; Organ; PTGS2 gene; Pathway interactions; Pharmaceutical Preparations; Phase; Physiological; Physiology; Plasma; Plastics; Population; Premature Birth; Premature Labor; Printing; Process; Prostaglandin-Endoperoxide Synthase; Protocols documentation; Reader; Reading; Reagent; Regulation; Reproducibility; Reproductive Medicine; Research; Sampling; Science; Shapes; Smooth Muscle; Smooth Muscle Myocytes; Staining method; Stains; Structure; Surface; System; Techniques; Testing; Time; Tissue Model; Tissues; Tocolysis; Tocolytic Agents; Toxic effect; Toxicity Tests; Translating; Tubulin; Uterine Contraction; Validation; Vimentin; base; body system; cell type; clinical effect; cost; cyclooxygenase 1; cytotoxicity; digital; efficacy testing; handheld mobile device; high throughput analysis; high throughput screening; in vitro Assay; in vitro Model; in vitro testing; in vivo; in vivo Model; monolayer; morphometry; myometrium; nanoparticle; novel; programs; protein expression; public health relevance; response; success; three-dimensional modeling; tool; two-dimensional; usability; uterine contractility; uterine smooth muscle cell

DESCRIPTION (provided by applicant): Several disorders in reproductive medicine are results of changes in smooth muscle contractile activity. Increased myometrial contraction can lead to preterm labor, which affects 12% of the US population. However, the mechanisms involved in the transition from uterine quiescence to contractility at the onset of labor are not well-known. As a result, the management of labor disorders such as preterm birth is poor, particularly tocolytic therapies, which could delay preterm labor, but have not been effectively proven and tested for this purpose. The slow progress in understanding myometrial contractility and tocolytic management of preterm labor can be attributed to the lack of faithful in vitro models of the myometrium, as well as the ability to efficiently screen tocolytic compounds in a high-throughput fashion. While in vivo models are used in the uterine contractility research, pronounced differences in how animals and humans give labor mean that pathological changes have different biological bases and responses to drugs. Beyond these intrinsic differences, animal models are time-consuming, costly, and ethically challenging. Alternatively, ex vivo human myometrial tissue are useful models for uterine contractility research. Yet, ex vivo models are not ideal for robust studies on uterine contractility research, as they suffer from ethical issues, sample inconsistencies, and scarcity. As a result, in vitro assays have been explored as cheaper and robust alternatives to study the efficacy of tocolytic substances to predict efficacy in humans, or as a compound screen before in vivo testing. The development of in vitro cell culture models and organ systems has greatly facilitated the study of gene expression and pathway regulation within human myometrial tissues, as well as identify and characterize target pathways. The success of these studies confirms the ability to study uterine contractility at the cellular level by examination of electrical conduction and protein expression. However, in general, in vitro cell culture models are limited by their accuracy, likely due to the fact that most in vitro testing is performed on two-dimensional (2D) glass or plastic surfaces, or organ bath systems that do not fully represent the native human myometrium environment. Specifically to uterine contractility research, while in vitro models allow for a myriad of metrics related to uterine activity to be monitored in a controlled environment, these models are prone to spontaneous activity involving both activating and spontaneous contractile mechanisms that require suppression of the activating mechanism to achieve consistency in results. Additionally, limitations still remain in 2D cell culture and in vitro uterine tissue models in studying tissue-lvel physiology and cellular pathways, respectively, resulting in data lacking context, detail, accuracy and mostly reproducibility. Given these limitations, this proposal looks toward three-dimensional (3D) models, which more accurately can represent the native tissue environment. Specifically, a recently explored assay, the BiO Assay, will be applied to contractility research as the C-BiO Assay. The basis of the C-BiO Assay is magnetic printing of cells. Cells are incubated with nontoxic magnetic nanoparticles that render the cells magnetic. Using ring-shaped magnets, the cells are then printed in 96-well plates into 3D rings, which close over time and at a rate that varies with compound concentration. The C-BiO Assay uses label-free metrics, so it does not require any reagents, dyes, or specialized equipment. Furthermore, data is gathered using a mobile device, which can be programmed to image whole plates at specific time points, avoiding the time-consuming imaging of individual wells under a microscope or reading plates on a plate reader, that is involved in 2D in vitro assays. Our hypothesis is that the C-BiO Assay will apply the benefits of 3D cell culture to an area of need, the lack of a faithful in vitro myometrial mode for contractility research, while being faster than other assay systems. In this Phase I proposal, the parameters of the C-BiO Assay will be optimized for high-throughput screening. Then, the assay will be compared to other 2D and 3D assays, and validated as a measure of smooth muscle contraction. This assay 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 compouns and their mechanisms of actions  Tools for high-throughput analysis that could significantly cut the time and cost of data collection The end result is an assay that mimics the myometrial structure and physiology, particularly smooth muscle contraction, and allows for high-throughput testing to efficiently screen tocolytic compounds for efficacy and toxicity. Aims Aim 1 - Optimization of the Magnetic Levitation and Printing of Myometrial 3D Cell Cultures for the C-BiO Assay Aim 2 - Validation of the 3D Myometrial C-BiO Assay.

描述（由申请人提供）：生殖医学中的几种疾病是平滑肌收缩活动变化的结果。肌层收缩增加可能导致早产，这影响了美国12％的人口。但是，在劳动开始时从子宫静止到收缩性的过渡涉及的机制并不众所周知。结果，诸如早产之类的劳动力障碍的管理很差，尤其是溶菌疗法，可能会延迟早产劳动，但尚未得到有效证明和测试。理解肌层收缩力和早产疗法管理的缓慢进展可以归因于缺乏忠实的子宫肌层体外模型，以及以高通量方式有效筛选溶血化合物的能力。尽管体内模型用于子宫收缩性研究，但动物和人类如何提供劳动的明显差异意味着病理变化具有不同的生物学基础和对药物的反应。除了这些内在差异之外，动物模型还耗时，昂贵且在道德上具有挑战性。另外，体内人肌层组织是子宫收缩性研究的有用模型。然而，由于遭受道德问题，样本不一致和稀缺性，因此体内模型并不是对子宫收缩性研究的强大研究的理想选择。结果，已经探索了体外测定法作为研究溶溶性物质预测人类疗效或体内测试之前的复合筛查的疗效的较便宜，可靠的替代方法。体外细胞培养模型和器官系统的开发极大地促进了人肌层组织内基因表达和途径调节的研究，并识别和表征了靶途径。这些研究的成功证实了通过检查电导传导和蛋白质表达在细胞水平上研究子宫收缩力的能力。 但是，通常，体外细胞培养模型受其准确性的限制，这可能是由于大多数体外测试是在二维（2D）玻璃或塑料表面上进行的，或者不完全代表人类肌层本地环境的器官浴系统。专门针对子宫收缩性研究，而体外模型允许无数的指标 这些模型与要在受控环境中监测的子宫活性相关，这些模型容易自发活动，涉及激活和自发的收缩机制，这些机制需要抑制激活机制以达到结果的一致性。另外，在研究组织层次生理学和细胞途径的2D细胞培养和体外子宫组织模型中仍然存在局限性，从而导致数据缺乏背景，细节，准确性 主要是可重复的。鉴于这些局限性，该建议将介绍三维（3D）模型，该模型可以更准确地代表天然组织环境。具体而言，最近探索的分析（生物测定法）将作为C-BIO分析应用于收缩性研究。 C-BIO分析的基础是细胞的磁打印。将细胞与使细胞磁性的无毒磁性纳米颗粒一起孵育。然后，使用环形磁铁将细胞在96孔板中印刷成3D环，随着时间的流逝，它们会随着复合浓度而变化。 C-BIO分析使用无标签的指标，因此不需要任何试剂，染料或专用设备。此外，可以使用移动设备收集数据，该设备可以在特定时间点进行编程，以避免在显微镜下的单个井的耗时成像或在板块读取器上读取板的时间，这些成像涉及2D体外测定法。我们的假设是，C-BIO分析将将3D细胞培养的好处应用于需求领域，缺乏忠实的体外肌层研究模式进行收缩性研究，同时比其他测定系统更快。在此阶段I建议中，将优化C-BIO分析的参数用于高通量筛选。然后，将测定与其他2D和3D测定法进行比较，并将其验证为平滑肌收缩的量度。该测定法将整合：能够快速打印3D细胞培养物，具有相关的细胞外矩阵实时和无标签的环闭合量化量化，这与细胞功能的能力相关，该能力与细胞功能相关的能力研究了特定组合的基础细胞毒性及其对高促进分析的作用机制的基础细胞毒性，以使高直接分析的时间和成本尤其降低了最终的结构，最终是一个最终的结构。肌肉收缩，并允许进行高通量测试，以有效筛选出溶溶性化合物的功效和毒性。 AIM AIM 1-用于C -BIO分析AIM 2的磁性悬浮和打印的磁悬浮和打印 - 对3D肌层C -BIO分析的验证。