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

光学分析是细胞电生理学的有力工具。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局限性是它们不能确定 电池中的绝对电压。目前的商业方法只报告电压的质的相对变化，& 没有关于绝对静息电位、舒张期电位或动作电位波幅的信息。前期研究 开发绝对电压报告的尝试一直未获成功。此外，目前可用的电压- 由于以下原因，敏感染料(VSD)的实验持续时间非常有限(通常为&lt；30分钟)：1)高洗脱率和 内化率，将它们从电活性细胞膜上移除；2)高光毒性， 减少测量可能的曝光时间；3)急性染料毒性，这限制了膜负载和 照明，导致小信号和低信噪比。 这一建议克服了开发和优化我们的新系统的这两个主要障碍，该系统结合了我们的 VSD&我们独特的、坚固耐用的光学和分析系统，可确定膜的绝对电位。我们的 集成定量光学电生理(QOEP)系统由我们的专利长效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，在 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，并减缓了褪色/ 内化率。这使他们有能力在实验中使用长达4小时。这种戏剧性的进步 革命性地改变了可以进行的实验类型。具体地说，较慢的内部化速度使 实验者校准VSD的时间，使测量的光强可以直接与 跨膜电位。新一代VSD的光谱特性和稳定性被结合在一起 随着电子和电路的进步，提高了信号灵敏度，并允许QOEP。染料性能与信号 加工过程因物种和器官/细胞类型而异。这些系统具有高度的细胞异质性， 相对于培养细胞的结缔组织。为了开发一个一致的系统，我们将优化我们的QOEP系统 特别适用于诱导多能干细胞来源(IPSCD)心脏的电合胞制剂 肌细胞。其目标是优化细胞系统(干细胞来源的心肌细胞)&制造 集成光学系统，使QOEP几乎可用于任何实验室。此转换将类似于 分子生物学试剂盒的出现使所有人都能获得复杂的分子生物技术。这个 长期的商业机会是心脏安全筛查，以确定新药的致心律失常潜力 干细胞来源的心肌细胞的候选者。我们的新系统有可能对 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 由传感器、染料、照明源和软件组成的系统，可用于第二阶段的测试版测试。 第二阶段，我们将为交钥匙商业系统开发软件、支持、打包和优化硬件。

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.