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: 8434994
• 负责人: Fabio Cerignoli
• 金额: $ 71.05万
• 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/8434994
• 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）：尚未报告高通量中个体心肌细胞的生理学。无法进行高吞吐物生理测量限制了许多基本和应用研究，包括在心脏毒性测试中使用干细胞衍生的心肌。当前的自动化心脏毒性测试的预测价值较差，因为它们使用了由单个通道（例如HERG）设计的肿瘤细胞系，并且在相对较晚的发育阶段，对很少的候选者保留了与生理相关的测试。这些模型的生物学相关性不佳，导致FDA批准之前甚至商业化后，候选药物的高失败率。我们从肌细胞中进行了自动记录以获取钙瞬变，但仍然受到电极设备进行起搏的限制，从而防止了96井格式以上的微型化。此外，可激发细胞中最相关的生理参数的动作势测量仍然保留为低吞吐量分析。我们提出了几种概念进步，通过开发一个基于细胞的小细胞光遗传步调装置来解决这些问题，以在自动化平台中对人类诱导的多能干细胞（HIPSC）衍生的心肌细胞进行高吞吐量分析，以用于细胞的细胞细胞细胞仪分析心肌细胞生理学的细胞细胞量表分析。我们还将通过荧光电压探针和录制后跟踪开发自动分割/分析动作电位（AP），以识别固定和免疫染色分析后的相同细胞。钙瞬态（CT）分析已经在先前的SBIR合同中开发，将与AP和录制后跟踪收敛，以生成对高吞吐量进行的所有这些终点的单细胞多参数测量。将使用不同的机制来改变AP以验证平台的药物进行广泛的评估。初步数据表明，表达光触发蛋白通道Ropopsin-2（CHR2）的稳定细胞系将电到偶发到心肌细胞，从而在不中断正常心肌细胞生理学的情况下进行光学控制的AP刺激。膜AP可以通过电压探针记录在心肌细胞中，并且适合图像分割分析。自动CT测量和HIPSC衍生的心肌细胞是测试参考药物的心脏毒性作用的有效模型。目的将推动使用荧光探针来响应刺激来测量作用电位，钙通量和细胞特征。心肌细胞生理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。该软件将将图像分割为单个单元格记录，因此所有测量结果和数据分析都将逐个单元格。将评估该格式为384和1536孔，以对每个单个数据点（例如，经过测试的化合物）进行数百个单元格进行筛选，从而在单个屏幕中允许在融资期结束时在单个筛选中吞吐量至数十万个数据点。通道开启器和阻滞剂将进行测试以验证平台。该平台将在基础研究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。