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

在即用型检测板中冷冻保存干细胞衍生的心肌细胞，以提高药物毒性测试的重现性

• 批准号: 10082253
• 负责人: Eli Fine
• 金额: $ 25.21万
• 依托单位: CURI BIO INC
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-11 至 2022-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10082253
• 关键词: Address; Adoption; Affect; Animal Experimentation; Behavior; Biological Assay; Biological Models; Biomimetics; Carbon Dioxide; Cardiac; Cardiac Myocytes; Cardiotoxicity; Cell Survival; Cells; Cellular Morphology; Chronic; Clinical; Clinical Trials; Cryopreservation; Cryopreserved Tissue; Data; Detection; Development; Devices; Disease; Drug Costs; Drug toxicity; Early Diagnosis; Economics; Electronics; Electrophysiology (science); Elements; Exposure to; Extravasation; Failure; Fluorescence; Freezing; Frequencies; Funding; Generations; Giant Cells; Heart; Human; In Vitro; Industry; Industry Standard; Isoproterenol; Letters; Liquid substance; Logistics; Manufacturer Name; Methods; Microelectrodes; Modification; Optics; Outcome; Pain; Patients; Pattern; Pharmaceutical Preparations; Phase; Phenotype; Physiological; Preparation; Price; Procedures; Process; Production; Protocols documentation; Publications; Quality Control; Reader; Reproducibility; Research Personnel; Robotics; Safety; Screening procedure; Shipping; Ships; Site; Source; Stress; Structure; System; Technology; Temperature; Testing; Time; Tissues; Toxic effect; Toxicity Tests; Translating; Work; atmospheric carbon dioxide; axion; base; cell motility; cell type; commercialization; cost; cryogenics; design; dofetilide; drug candidate; drug development; drug discovery; experience; experimental study; falls; health care delivery; human subject; human tissue; improved; in vitro Model; induced pluripotent stem cell; luminescence; microscopic imaging; monolayer; multi-electrode arrays; nanopattern; next generation; novel strategies; novel therapeutics; patient population; pre-clinical; preclinical toxicity; predictive modeling; prevent; process optimization; response; screening; stem cells; tool; validation studies; wasting

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.

项目总结/摘要 近90%的开发中药物未能进入市场。许多失败是由于心脏毒性引起的。 在少数值得注意的情况下，一些药物通过了临床前筛选和临床试验，但却被从临床试验中删除。 一旦在大量患者人群中发现毒性作用，这些失败代表了一个巨大的 这是废物的来源，并构成将单一药物推向市场的约20亿美元成本的重要部分。 因此，FDA现在要求所有药物在进行体外心脏毒性试验之前， 人类这导致了一个重要的和不断增长的市场的工具和技术，使早期检测 在接触到病人之前，然而，目前的筛查方法无法预测 药物将在体内发挥作用;事实上，迫切需要更多的预测模型系统。人类 诱导多能干细胞衍生的心肌细胞（iPSC-CM）是体外临床前研究的有吸引力的模型。 毒性筛选;它们来自人体组织，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（特别是高度流行的多功能测定板）是困难的。 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可变性 和低的站点间重现性。药物发现行业及其监管机构意识到iPSC-CM的潜力 用于早期心脏毒性筛查，但也要了解目前其使用存在重大限制 在药物开发过程中由实验变异性引起的。为了解决这个问题，目前一些公司 提供内部已经有细胞的“检测就绪”平板，然后在环境条件下将其运送给客户。 然而，细胞通常不会长时间暴露在环境条件下，这引起了人们的质疑。 这种方法的科学有效性-以及无法储存易腐产品的问题， 制造和客户使用。然而，对当前一代环境测定的高需求- 准备好的盘子（见支持信）清楚地表明，它们代表了减少 药物开发的成本和浪费。NanoSurface Biomedical，Inc.旨在开发下一代“检测- 通过向客户运送含有iPSC-CM单层的低温冷冻测定板，我们 假设最近在冷冻保存其他细胞类型单层方面的进展可以成功地转化为 并针对iPSC-CM进行了优化。我们将首先集中展示iPSC-CM和测定板保持 作为单层低温储存后的活力和完整性（目标1），然后进行概念验证研究 显示iPSC-CM功能不受冷冻/解冻过程的不利影响（目的2）。后续 第二阶段的商业化工作将侧重于优化冷冻和解冻过程， 商业化生产，充分表征iPSC-CM板的结构和功能表型， 解冻，确定一套关键的生理指标，将用于质量控制，设计和建设 辅助硬件设备，可在解冻过程中在客户现场实现最佳再现性。