Optogenetic Multiparametric Assay for HT Cardiotoxicity Testing

HT 心脏毒性测试的光遗传学多参数测定

• 批准号: 8656563
• 负责人: Fabio Cerignoli
• 金额: $ 52.1万
• 依托单位: VALA SCIENCES, INC.
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-04-01 至 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8656563
• 关键词: Action Potentials; Affect; Algorithms; Antibodies; Applied Research; Basic Science; Biological; Calcium; Cardiac; Cardiac Myocytes; Cardiotoxicity; Cardiovascular Diseases; Cell Line; Cell membrane; Cells; Characteristics; Clinical; Clinical Trials; Coculture Techniques; Collection; Computer software; Computers; Contracts; Data; Data Analyses; Development; Devices; Electrodes; Electrophysiology (science); Engineering; Environment; Evaluation; Failure; Fiber Optics; Fluorescent Probes; Funding; Generations; Heart; Heart Atrium; Human; Image; Image Analysis; Immunofluorescence Immunologic; Individual; International; Ion Channel; Kinetics; Laboratories; Libraries; Light; Link; Measurement; Measures; Medical Research; Membrane; Methodology; Methods; Microscope; Miniaturization; Modeling; Monitor; Muscle Cells; Neonatal; Nuclear; Pharmaceutical Preparations; Phase; Physiological; Physiology; Population; Predictive Value; Problem Solving; Process; Proteins; Rattus; Recording of previous events; Records; Regenerative Medicine; Reporting; Research Institute; Safety; Sampling; Science; Small Business Innovation Research Grant; Speed; Staging; Staining method; Stains; Stem cells; Subgroup; System; Techniques; Technology; Testing; Tumor Cell Line; United States National Institutes of Health; Validation; Ventricular; assay development; base; charge coupled device camera; commercialization; cost; design; drug candidate; drug development; drug testing; high throughput analysis; high throughput screening; induced pluripotent stem cell; instrument; miniaturize; optogenetics; prevent; protein expression; prototype; release of sequestered calcium ion into cytoplasm; research and development; research study; response; safety testing; sample fixation; screening; segmentation Image analysis; small molecule; small molecule libraries; software development; stable cell line; voltage

DESCRIPTION (provided by applicant): Monitoring physiology of individual cardiomyocytes in high throughput has not been reported. The inability to perform high throughput physiological measurements limits many basic and applied studies, including the use of stem cell derived cardiomycoytes in cardiotoxicity testing. Current automated cardiotoxicity tests have poor predictive value because they use tumor cell lines engineered with single channels (e.g. hERG), and physiologically relevant tests are reserved for few candidates during the relatively late stages of development. The poor biological relevance of these models contributes to the high failure rate of drug candidates before FDA approval and even after commercialization. We have automated recording from myocytes for Calcium Transients, but are still limited by use of electrode devices for pacing that prevents miniaturization beyond 96- well format. Furthermore, Action Potential measurement, the most relevant physiological parameter in excitable cells, is still reserved to low throughput analysis. We propose several conceptual advances to solve these problems by developing a miniaturized, cell-based optogenetic pacing device for high throughput analysis of human Induced Pluripotent Stem Cell (hIPSC)-derived cardiomyocytes in an automated platform for cell-by-cell cytometric analysis of cardiomyocyte physiology. We will also develop automatic segmentation/analysis of Action Potentials (AP) through fluorescent voltage probes and post-recording tracking to identify the same cells after fixation and immunostaining analysis. Calcium Transient (CT) analysis, already developed in a previous SBIR contract, will converge with AP and post-recording tracking to generate single cell multiparametric measurement of all these endpoints conducted in High Throughput. Extensive evaluation will be conducted with drugs that alter AP through different mechanisms to validate the platform. Preliminary data show that stable cell lines expressing the light-triggered protein Channelrhodopsin-2 (ChR2) will electrically couple to cardiomyocytes, allowing optically controlled stimulation of AP without disruption of normal cardiomyocyte physiology. Membrane AP can be recorded in cardiomyocytes through voltage probes and are suitable to image segmentation analysis. Automatic CT measurement and hIPSC-derived cardiomyocytes are an effective model to test cardiotoxic effects of reference drugs. The Aims will advance the use of fluorescent probes to measure action potential, calcium flux and cell characteristics in response to the stimulation. Cardiomyocyte physiology will be quantified by image analysis software that records and analyzes single-cell AP and CT in relation to cardiac subtype or specific protein expression. The software will segment the images into single cell recordings, thus all measurements and data analysis will be on a cell-by- cell basis. The format will be evaluated for 384- and 1536-well to conduct screening on hundreds of cells per individual data point (e.g. compound tested), allowing throughput of tens to hundreds of thousands of datapoints in a single screen by the end of the funding period. Channel openers and blockers will be tested to validate the platform. The platform will find applications in basic and applied research, including regenerative medicine research and drug development/safety testing.

描述（由申请人提供）：尚未报道以高通量监测单个心肌细胞的生理学。无法进行高通量生理测量限制了许多基础和应用研究，包括在心脏毒性测试中使用干细胞衍生的心肌细胞。目前的自动化心脏毒性测试的预测价值很差，因为它们使用的是单通道工程设计的肿瘤细胞系（例如 hERG），并且生理相关测试只针对开发相对较晚阶段的少数候选者。这些模型的生物学相关性较差，导致候选药物在 FDA 批准之前甚至商业化之后的失败率很高。我们已经自动记录了肌细胞的钙瞬变，但仍然受到使用电极装置进行起搏的限制，这阻碍了超过 96 孔格式的小型化。此外，动作电位测量（可兴奋细胞中最相关的生理参数）仍然保留用于低通量分析。我们提出了一些概念性进展来解决这些问题，通过开发一种小型化的、基于细胞的光遗传学起搏装置，在对心肌细胞进行逐个细胞细胞计数分析的自动化平台中，对人类诱导多能干细胞（hIPSC）衍生的心肌细胞进行高通量分析生理。我们还将通过荧光电压探针和记录后跟踪开发动作电位（AP）的自动分割/分析，以在固定和免疫染色分析后识别相同的细胞。钙瞬态 (CT) 分析已在之前的 SBIR 合同中开发，将与 AP 和记录后跟踪融合，以生成在高吞吐量下进行的所有这些端点的单细胞多参数测量。将通过不同机制改变 AP 的药物进行广泛的评估，以验证该平台。初步数据显示，表达光触发蛋白视紫红质通道蛋白-2 (ChR2) 的稳定细胞系将与心肌细胞电耦合，从而可以在不破坏正常心肌细胞生理学的情况下对 AP 进行光控刺激。可以通过电压探针记录心肌细胞内的膜AP，适合图像分割分析。自动CT测量和hIPSC来源的心肌细胞是测试参考药物心脏毒性作用的有效模型。该目标将推进荧光探针的使用，以测量动作电位、钙通量和细胞对刺激的反应特征。心肌细胞生理学将通过图像分析软件进行量化，该软件记录和分析与心脏亚型或特定蛋白质表达相关的单细胞 AP 和 CT。该软件将图像分割成单细胞记录，因此所有测量和数据分析都将逐个细胞进行。该格式将针对 384 孔和 1536 孔进行评估，以对每个数据点的数百个细胞进行筛选（例如测试的化合物），从而在资助期结束时在一次筛选中实现数万至数十万个数据点的吞吐量。将测试渠道开放者和阻碍者以验证该平台。该平台将在基础和应用研究中找到应用，包括再生医学研究和药物开发/安全测试。