Cryopreserving Stem Cell-Derived Cardiomyocytes in Ready-to-use Assay Plates for Improved Reproducibility of Drug Toxicity Testing

项目摘要/摘要 近90%正在研发的药物未能上市。其中许多失败是由于心脏毒性造成的。 在少数值得注意的案例中，一些药物通过了临床前筛选和临床试验，结果却被从 一旦在大量患者群体中发现毒副作用，就会进入市场。这些失败代表着一个巨大的 这是废物的来源，而且不能构成将一种药物推向市场的约20亿美元成本的一部分。 因此，FDA现在要求所有药物在进行体外心脏毒性测试之前，必须先进行心脏毒性测试。 人类。这导致了对能够更早检测的工具和技术的显著fiCan和不断增长的市场 在接触病人之前的毒性反应。然而，目前的筛查方法不能预测如何 药物将在体内发挥作用；事实上，迫切需要更具预测性的模型系统。人类 诱导多能干细胞来源的心肌细胞(IPSC-CMS)是一种有吸引力的体外基础研究模型 毒性筛选；它们来自人类组织，有可能减少对动物的需求 实验。然而，目前IPSC-CM测定板的制备(特别是非常流行的多孔板) 电极阵列板)在技术上具有挑战性，这导致更高的操作员诱导的实验变异性 以及站点到站点的低重复性。药物发现行业及其监管机构认识到IPSC-CMS的潜力 对于早期心脏毒性筛查，但也要了解目前他们的使用存在显著的fi限制 在药物开发过程中引起的实验变异性。为了解决这个问题，目前一些公司 提供已有细胞的“可用于分析的”培养板，然后在环境条件下运往客户手中。 然而，电池通常不会暴露在环境条件下这么长时间--这令人怀疑 该方法科学的fic有效性--以及不能存储易腐烂的产品在 制造和客户使用。然而，对当前一代环境检测的高需求- 准备好的车牌(见支持函)清楚地表明，它们代表着一个重要的fiCan机会 药物开发中的成本和浪费。NanoSurface Biomedical，Inc.致力于开发下一代“分析-- 通过向客户发货含有IPSC-CM单层的低温冷冻试纸板，我们可以使用“Ready”技术。我们 假设其他细胞类型的冷冻保存单分子层的最新进展可以成功地转化 并针对IPSC-CMS进行了优化。我们将首先集中展示fi-CMS和分析板保持 作为单层冷藏后的生存能力和完整性(目标1)，然后进行概念验证研究 表明IPSC-CM功能没有受到冻结/解冻进程的不利影响(目标2)。后续 第二阶段的商业化工作将重点放在优化冻结和解冻过程，以扩大和 商业生产，充分表征IPSC-CM板的结构和功能表型 解冻，确定一组将用于质量控制的关键生理指标，以及设计和建造 辅助硬件设备，可在解冻过程中实现客户现场的最佳重现性。

PROJECT SUMMARY / ABSTRACT Nearly 90% of drugs under development fail to reach the market. Many of these failures occur due to cardiotoxicity. In a few notable cases, some drugs pass preclinical screens and clinical trials, only to be removed from the market once toxic effects are discovered in large patient populations. These failures represent a tremendous source of waste and constitute a significant part of the ~$2 billion cost of bringing a single drug to market. Consequently, the FDA now mandates that all drugs undergo in vitro cardiotoxicity testing before being tested in humans. This has led to a significant and growing market for tools and technologies that enable earlier detection of toxic effects before exposure to patients. However, current screening methods fall short of predicting how a drug will behave in the body; indeed there is a pressing need for more predictive model systems. Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) are an attractive model for in vitro preclinical toxicity screening; they are derived from human tissue and have the potential to reduce the need for animal experimentation. However, at present, preparation of iPSC-CM assay plates (particularly the highly popular multi- electrode array plate) is technically challenging, which leads to higher operator-induced experimental variability and low site-to-site reproducibility. The drug discovery industry and its regulators realize the potential of iPSC-CMs for early cardiotoxicity screening, but also understand that there are currently significant limitations to their use in the drug development process caused by experimental variability. To address this, some companies currently provide “assay-ready” plates with cells already inside, which are then shipped to customers at ambient conditions. However, cells are not normally exposed to ambient conditions for such long periods of time—calling into question the scientific validity of that approach—and the inability to store the perishable product causes pain points in manufacturing and customer use. Nevertheless, the high demand for the current generation of ambient assay- ready plates (see Letters of Support) makes it clear that they represent a significant opportunity for reducing cost and waste in drug development. NanoSurface Biomedical, Inc. aims to develop the next generation of “assay- ready” technology by shipping customers cryogenically frozen assay plates containing iPSC-CM monolayers. We hypothesize that recent advances in cryopreserving monolayers of other cell types can be successfully translated and optimized for iPSC-CMs. We will first focus on demonstrating that iPSC-CMs and assay plates maintain viability and integrity after cryogenic storage as a monolayer (Aim 1), then conduct proof-of-concept studies showing that the iPSC-CM function is not adversely affected by the freeze/thaw process (Aim 2). Subsequent Phase 2 commercialization efforts will focus on optimizing the freezing and thawing process for scaling and commercial production, fully characterizing structural and functional phenotypes of the iPSC-CM plates after thaw, identifying a set of key physiological metrics that will be used for quality control, and designing and building ancillary hardware devices that will allow optimal reproducibility at the customer site during the thawing process.