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

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.PROJECT NARRATIVE In this project, the company will develop methods to produce pre-plated cardiac tissues cryopreserved in assay- ready plates, which can be used to preclinically assess how the human heart will react to drugs. We anticipate that the reduced cost, ease of workflow, and improved repeatability and reproducibility of cyropreserved plates will greatly enhance the utility of cardiac tissues in providing more predictive safety and efficacy data than currently available using existing technologies. This product has great potential to dramatically reduce the cost of drug development, improve clinical outcomes, and save lives across a wide spectrum of diseases and conditions.