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。子宫肌肌收缩加剧可导致早产，这影响了12%的美国人口。然而，从子宫静止到分娩时收缩的机制尚不清楚。因此，对早产等劳动障碍的管理很差，特别是可延迟早产的抗胎压治疗，但尚未为此目的得到有效证明和测试。在了解子宫肌层收缩性和早产的妊娠管理方面进展缓慢，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妊娠化合物的能力。虽然子宫收缩性研究采用体内模型，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。除了这些内在差异之外，动物模型耗时、昂贵，而且在道德上具有挑战性。另外，离体人子宫肌组织是子宫收缩性研究的有用模型。然而，由于存在伦理问题、样本不一致和稀缺性，离体模型并不适合用于子宫收缩性研究。因此，已经探索了体外测定法作为更便宜和可靠的替代方法来研究促孕物质的功效，以预测其在人体内的功效，或作为体内试验前的复合筛选。体外细胞培养模型和器官系统的发展，极大地促进了人类肌组织内基因表达和通路调控的研究，以及靶通路的识别和表征。这些研究的成功证实了在细胞水平上通过检查电传导和蛋白质表达来研究子宫收缩能力的能力。