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（由申请方提供）：生殖医学中的几种疾病是平滑肌收缩活性变化的结果。子宫肌层收缩加剧可导致早产，影响12%的美国人口。然而，在分娩开始时子宫从静止到收缩的转变所涉及的机制并不清楚。因此，对早产等分娩障碍的管理很差，特别是保胎治疗，这可能会延迟早产，但尚未被有效地证明和测试。在了解子宫肌层收缩性和宫缩抑制管理早产的进展缓慢，可以归因于缺乏忠实的子宫肌层的体外模型，以及能够有效地筛选宫缩抑制化合物在高通量的方式。虽然在子宫收缩力研究中使用了体内模型，但动物和人类分娩方式的显著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。除了这些内在的差异，动物模型是耗时的，昂贵的，道德上的挑战。或者，离体人子宫肌层组织是子宫收缩力研究的有用模型。然而，离体模型对于子宫收缩力研究的稳健研究并不理想，因为它们存在伦理问题，样品不一致和稀缺性。因此，体外试验已被探索为更便宜和稳健的替代方案，以研究保胎物质的功效来预测人体功效，或作为体内试验前的化合物筛选。体外细胞培养模型和器官系统的发展极大地促进了人类子宫肌层组织内基因表达和通路调控的研究，以及识别和表征靶通路。这些研究的成功证实了通过检查电传导和蛋白质表达在细胞水平上研究子宫收缩力的能力。 然而，一般来说，体外细胞培养模型受到其准确性的限制，这可能是由于大多数体外试验是在二维（2D）玻璃或塑料表面或器官浴系统上进行的，这些表面不能完全代表天然的人类子宫肌层环境。特别是子宫收缩力研究，而体外模型允许无数的指标 与在受控环境中监测的子宫活动相关，这些模型倾向于涉及激活和自发收缩机制的自发活动，其需要抑制激活机制以实现结果的一致性。此外，2D细胞培养和体外子宫组织模型在分别研究组织水平生理学和细胞通路方面仍然存在局限性，导致数据缺乏背景、细节、准确性和可操作性。 主要是可重复性。考虑到这些限制，该提议着眼于三维（3D）模型，其可以更准确地表示天然组织环境。具体而言，最近开发的测定法，生物测定法，将作为C-BiO测定法应用于收缩性研究。C-BiO检测的基础是细胞的磁性打印。细胞与无毒的磁性纳米颗粒一起孵育，使细胞具有磁性。使用环形磁铁，将细胞在96孔板中打印成3D环，这些环会随着时间的推移而闭合，并且速度会随着化合物浓度的变化而变化。C-BiO Assay使用无标记指标，因此不需要任何试剂、染料或专用设备。此外，使用移动终端收集数据，该移动终端可被编程为在特定时间点对整个板进行成像，避免了在显微镜下对单个威尔斯孔进行耗时成像或在读板器上对板进行阅读，这涉及2D体外测定。我们的假设是，C-BiO检测将3D细胞培养的优势应用于需要的领域，缺乏用于收缩性研究的忠实的体外子宫肌层模式，同时比其他检测系统更快。在本I期提案中，将针对高通量筛选优化C-BiO Assay的参数。然后，将该测定与其他2D和3D测定进行比较，并验证其作为平滑肌收缩的测量。该检测试剂盒将整合：  能够快速打印具有相关细胞外基质的3D细胞培养物  实时和无标记定量环闭合，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，可以显著减少数据收集的时间和成本最终的结果是一种模拟子宫肌层结构和生理学，特别是平滑肌收缩的测定，并允许高通量测试，以有效筛选宫缩抑制化合物的功效和毒性。目的目的1 -用于C-BiO测定的子宫肌层3D细胞培养物的磁悬浮和打印的优化目的2 - 3D子宫肌层C-BiO测定的验证。