A System to Optically Determine the Absolute Membrane Potential in Human iPSCD Cardiac Myocytes

光学测定人 iPSCD 心肌细胞绝对膜电位的系统

• 批准号: 10081467
• 负责人: Anthony John Costantino
• 金额: $ 25万
• 依托单位: CYTOCYBERNETICS, INC.
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-09 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10081467
• 关键词: Action Potentials; Acute; Algorithms; Biological; Biological Assay; Brugada syndrome; Calibration; Cardiac; Cardiac Electrophysiologic Techniques; Cardiac Myocytes; Cell membrane; Cells; Chemicals; Complex; Computer software; Connective Tissue; Cultured Cells; DNA Sequence Alteration; Data; Detection; Development; Devices; Disease; Doctor of Philosophy; Dyes; Electrodes; Electronics; Electrophysiology (science); Endothelium; Fibroblasts; Fluorescence; Generations; Genetic study; Goals; Health; Heterogeneity; Hour; Human; Image; In Vitro; Industry; Knowledge; Legal patent; Lighting; Manuals; Measurement; Measures; Mechanics; Membrane; Membrane Potentials; Methods; Microscope; Modeling; Molecular; Molecular Biology; Monitor; Nature; Noise; Optical Methods; Optics; Organ; Performance; Pharmaceutical Preparations; Phase; Phototoxicity; Postdoctoral Fellow; Preparation; Property; Protocols documentation; Quality Control; Recording of previous events; Reporting; Reproducibility; Research; Research Personnel; Rest; Safety; Signal Transduction; Source; Standardization; Study Subject; Syndrome; System; Techniques; Testing; Time; Toxic effect; Validation; Work; base; cell type; design; detector; drug candidate; drug development; electrical measurement; experimental study; fluorescence microscope; induced pluripotent stem cell; light intensity; medication safety; novel; novel therapeutics; optical imaging; optogenetics; patch clamp; pre-clinical; quantum; screening; sensor; signal processing; software development; stem cells; systems research; temporal measurement; tool; virtual; voltage; voltage sensitive dye

Optical assays are a powerful tool in cellular electrophysiology. However, currently-available approaches have not reached their full potential. The major limitation of existing optical systems is that they are unable to determine the absolute voltage in the cell. Current commercial approaches report only qualitative relative changes in voltage, & there is no information on absolute resting potential, diastolic potential, or action potential amplitude. Prior research attempts to develop absolute voltage reports have been unsuccessful. In addition, currently available voltage- sensitive dyes (VSDs) offer a very limited experimental duration (typically < 30 min) dyes due to: 1) high washout & internalization rates, which removes them from the electrically active cell membrane; 2) high photo-toxicity, which reduces the possible exposure time for measurements; & 3) acute dye toxicity, which limits membrane loading & illumination, resulting in small signals & low signal to noise ratio. This proposal overcomes these 2 major obstacles to develop & optimize our novel system which combines our VSDs & our unique & robust optical & analytical system which determines absolute membrane potential. Our integrated quantitative Optical Electrophysiology (qOEP) system consists of our patented long lasting VSDs, optimized experimental protocols, optical detection system, & analytical software. Our VSDs, which operate in the red/NIR spectral range, have reduced acute chemical & photo-toxicity, increased sensitivity, & slower washout/ internalization rate. This gives them the ability to be used in experiments up to 4 hours. This dramatic improvement revolutionizes the types of experiment which can be performed. Specifically, slower internalization rate gives the experimenter time to calibrate the VSD, so that the measured light intensity can be directly correlated with transmembrane potential. The spectral properties & stability of this new generation of VSDs has been combined with advances in electronics & circuitry that increase signal sensitivity & allow for qOEP. Dye performance & signal processing are species & organ/cell type-specific. These systems have high degrees of cellular heterogeneity & connective tissue relative to cultured cells. To develop a consistent system we will optimize our qOEP system specifically for work with electrically syncytial preparations of induced pluripotent stem cell derived (IPSCD) cardiac myocytes. The goal is to optimize a cell system (stem cell derived cardiac myocytes) & the dyes to make an integrated optical system that makes qOEP available to almost any lab. This transformation will be similar to the way that the advent of molecular biology kits made complex molecular biological techniques accessible to all. The long term commercial opportunity is in cardiac safety screening to determine the arrhythmogenic potential of new drug candidates in stem cell derived cardiac myocytes. Our novel system has the potential to have significant impact in both the financial & human health aspects of drug development. Successful completion of Phase I will result in a system consisting of sensors, dyes, illumination sources, & software that can be used for beta testing in Phase II. In Phase II we will develop software, support, packaging & optimized hardware for a turn-key commercial system.

光学测定是细胞电生理学的强大工具。然而，目前可用的方法还没有 充分发挥了他们的潜力。现有光学系统的主要限制是它们无法确定 电池中的绝对电压。当前的商业方法仅报告电压的定性相对变化，& 没有关于绝对静息电位、舒张电位或动作电位幅度的信息。先前的研究 开发绝对电压报告的尝试并未成功。此外，目前可用的电压 敏感染料 (VSD) 的实验持续时间非常有限（通常 < 30 分钟），原因是：1) 高冲洗率和 内化率，将它们从电活性细胞膜上去除； 2）高光毒性， 减少测量可能的曝光时间； & 3) 急性染料毒性，限制膜负载 & 照明，导致小信号和低信噪比。 该提案克服了这两个主要障碍来开发和优化我们的新颖系统，该系统结合了我们的 VSD 和我们独特且强大的光学和分析系统可确定绝对膜电位。我们的 集成定量光学电生理学 (qOEP) 系统由我们获得专利的长效 VSD 组成， 优化的实验方案、光学检测系统和分析软件。我们的 VSD 运行于 红色/近红外光谱范围，降低了急性化学和光毒性，提高了灵敏度，并减缓了冲刷/ 内部化率。这使它们能够用于长达 4 小时的实验。这种戏剧性的进步 彻底改变了可以进行的实验类型。具体来说，较慢的内化速率给出了 实验者校准 VSD 的时间，以便测量的光强度可以直接与 跨膜电位。新一代 VSD 的光谱特性和稳定性得到了结合 电子和电路方面的进步提高了信号灵敏度并允许 qOEP。染料性能和信号 处理是特定于物种和器官/细胞类型的。这些系统具有高度的细胞异质性& 结缔组织相对于培养细胞。为了开发一致的系统，我们将优化我们的 qOEP 系统 专门用于诱导多能干细胞衍生 (IPSCD) 心脏的电合胞制剂 肌细胞。目标是优化细胞系统（干细胞衍生的心肌细胞）和染料以制造 集成光学系统使 qOEP 几乎适用于任何实验室。这种转变将类似于 分子生物学试剂盒的出现使所有人都可以使用复杂的分子生物学技术。这 长期的商业机会在于心脏安全筛查，以确定新药的致心律失常潜力 干细胞衍生的心肌细胞的候选者。我们的新颖系统有可能对以下领域产生重大影响： 药物开发的财务和人类健康方面。第一阶段的成功完成将导致 系统由传感器、染料、照明源和软件组成，可用于第二阶段的 beta 测试。在 第二阶段，我们将为交钥匙商业系统开发软件、支持、包装和优化硬件。