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性变化的结果。子宫肌层收缩加剧可能导致早产，影响 12% 的美国人口。然而，分娩开始时从子宫静止状态转变为收缩状态的机制尚不清楚。因此，对早产等分娩障碍的管理很差，特别是安胎疗法，这可能会延迟早产，但尚未为此目的得到有效证明和测试。对早产的子宫肌层收缩性和保胎管理的理解进展缓慢，可归因于缺乏可靠的子宫肌层体外模型，以及以高通量方式有效筛选保胎化合物的能力。虽然子宫收缩力研究使用体内模型，但动物和人类分娩方式的显着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、昂贵且在伦理上具有挑战性。另外，离体人类子宫肌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来说并不理想，因为它们存在伦理问题、样本不一致和稀缺性。因此，体外测定已被探索作为更便宜和可靠的替代方案来研究保胎物质的功效以预测对人类的功效，或作为体内测试之前的化合物筛选。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，以及目标通路的识别和表征。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，这可能是因为大多数体外测试是在二维 (2D) 玻璃或塑料表面或不能完全代表细胞的器官浴系统上进行的。人类原生子宫肌层环境。特别针对子宫收缩力研究，而体外模型允许多种指标 与在受控环境中监测的子宫活动相关，这些模型容易发生自发活动，涉及激活机制和自发收缩机制，需要抑制激活机制以实现结果的一致性。此外，二维细胞培养和体外子宫组织模型在研究组织级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、细节和准确性 以及主要的可重复性。鉴于这些限制，该提案着眼于三维 (3D) 模型，它可以更准确地表示天然组织环境。具体来说，最近探索的一种检测方法 BiO 检测将作为 C-BiO 检测应用于收缩性研究。 C-BiO 测定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。然后使用环形磁铁将细胞在 96 孔板中打印成 3D 环，随着时间的推移，这些环会以随化合物浓度变化的速度闭合。 C-BiO 测定使用无标记指标，因此不需要任何试剂、染料或专用设备。此外，使用移动设备收集数据，该移动设备可以编程为在特定时间点对整个板进行成像，从而避免在显微镜下对单个孔进行成像或在读板器上读取板，这在体外 2D 中涉及化验。我们的假设是，C-BiO 测定将 3D 细胞培养的优势应用于需要的领域，即缺乏用于收缩性研究的可靠体外肌层模式，同时比其他测定系统更快。在此第一阶段提案中，C-BiO 测定的参数将针对高通量筛选进行优化。然后，该检测将与其他 2D 和 3D 检测进行比较，并作为平滑肌收缩的测量方法进行验证。该测定将集成： 快速打印具有相关细胞外基质的 3D 细胞培养物的能力 与细胞功能相关的环闭合的实时无标记定量 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最终结果是模拟子宫肌层结构和生理学，特别是平滑肌收缩的测定，并允许进行高通量测试以有效筛选保胎化合物的功效和毒性。目标 目标 1 - 优化磁悬浮和打印用于 C-BiO 测定的子宫肌层 3D 细胞培养物 目标 2 - 验证 3D 子宫肌层 C-BiO 测定。