An in vitro electrophysiology system for high-throughput measurement of cardiomyocyte action potential

摘要 自1950年以来，心脏病一直是美国人的头号死因。此外，心血管安全 责任，特别是导致心律失常的风险，仍然是药物开发中高流失率的主要原因。为 对心脏功能障碍的机制和潜在治疗方法的研究，一直有来自 业界和学术界一致支持高质量、高通量的心脏动作电位(AP)分析，这是一种强大的 心功能不全的生物标志物。不幸的是，现有的测量AP的技术，包括电学和 光学方法都受到严重的限制，在技术上具有挑战性和/或大规模执行的成本很高。 人类来源的心肌细胞的严重异质性进一步加剧了这些局限性。 这项第一阶段的提案旨在开发一种支持高通量的新型体外电生理系统 使用人源性心肌细胞测定细胞内AP(IAP)。更具体地说，我们寻求 证明即使在电生理学方面的专业知识有限的研究人员也可以执行高通量 IAP分析，包括数据采集和分析。 这种电生理系统的核心是斯坦福大学开发的3D纳米电极阵列(NEA)，该阵列 弥合细胞内和细胞外电生理之间的差距，实现高质量和可扩展 许多单个心肌细胞的IAP记录。基于这项NEA技术，Cyion Technologies拥有 之前开发的第一代电生理系统(文森特)记录了高达60%的IAP信号 在单孔NEA底物中分离的心肌细胞。在这一成功的基础上，在本提案中，我们寻求开发一种 额外的“平板阅读器”选项，使用96孔NEA底物测量672个单个心肌细胞。我们会 还在用户软件中实施软件算法，以实现对IAP信号的自动化、批量分析，这是必要的 用于更高吞吐量的分析。

Summary Heart disease has been the #1 cause of fatality in the United States since 1950. Additionally, cardiovascular safety liability especially pro-arrhythmic risk remains a leading cause for the high attrition rate in drug development. For investigation of mechanisms and potential therapies for cardiac dysfunction, there has been strong demand from industry and academic alike for high quality, high-throughput assay of cardiac action potential (AP), a powerful biomarker for cardiac dysfunction. Unfortunately, existing techniques for measurement of AP, including electrical and optical methods, all suffer from severe limitations and are technically challenging and/or costly to execute at scale. These limitations are further compounded by the profound heterogeneity of human-derived cardiomyocytes. This Phase I proposal aims to develop a novel in vitro electrophysiology system that supports high-throughput measurement of intracellular AP (iAP) using human-derived cardiomyocytes. More specifically, we seek to demonstrate the feasibility for researcher even with limited expertise in electrophysiology to perform high-throughput iAP assay that includes data acquisition and analysis. Central to this electrophysiology system is a 3D nanoelectrode array (NEA) developed at Stanford University, which bridges the gap between intracellular and extracellular electrophysiology and enables high-quality and scalable recordings of iAP from many individual cardiomyocytes. Based on this NEA technology, Cyion Technologies has previously developed a first-generation electrophysiology system (Vincent) that records iAP signals from up to 60 discrete cardiomyocytes in a single-well NEA substrate. Built on this success, in this proposal we seek to develop an additional “plate-reader” option that measures 672 individual cardiomyocytes using a 96-well NEA substrate. We will also implement a software algorithm in user software for automated, batch analysis of iAP signals, which is necessary for higher-throughput assay.