279 Generation, high throughput screening, and biobanking of human iPSC-derived cardiac spheroids 11 INTRODUCTION The discovery of human-induced pluripotent stem cells (hiPSCs) offered unprecedented opportunities to study human development and disease at the cellular level. Over the past decade, using developmental lessons, various protocols were established to ensure the efficient differentiation of hiPSCs into cardiomyocytes (CMs)[1–4]. hiPSC-derived cardiomyocytes (hiPSC-CMs) can serve as a resource for modeling genetically inheritable cardiovascular diseases (CVDs), testing cardiac safety for new drugs, and cardiac regenerative strategies[5–8]. Despite directed cardiac differentiation of hiPSCs, indefinite cardiomyocyte numbers remain a challenge in the cardiac field since matured hiPSC-CMs generally are non-proliferative, and primary human cells are not available in high quantities. Recently, we described that concomitant Wnt signaling activation with low cell-density culture resulted in a massive proliferative response (up to 250 fold) of hiPSC-CMs[9,10]. This cost-effective strategy for the massive expansion of hiPSC-CMs via serial passaging in culture flask format facilitates standardization and quality control in large batches of functional hiPSC-CMs. Additionally, to keep up with the demand for large batches of hiPSC-CMs from various donors, the biobanking of hiPSC-CMs has been described[10]. However, cardiomyocyte monolayers seeded in these standard culture dishes are not representative of the complex 3D structure present in the heart. Moreover, the immaturity of hiPSC-CMs has remained an obstacle, thus falling short in mimicking the biological and physiological phenotype of the in vivo cardiovascular environment. Novel 3D in vitro models have been developed where hiPSC-CMs show closer physiological behavior such as self-organization processes[11,12], ECM remodeling[13], enhanced maturation[14–16], and synchronized contraction[17–19]. Hereto, 3D models have been utilized for drug discovery, drug cardiotoxicity testing, disease modeling, regenerative therapies, and even the first clinical trials[20–24]. One of the most used models is the fibrin-based engineered heart tissue (EHT), which exhibits a tissue-like arrangement and cardiac contractility[13,17,25]. Previously, we showed that EHTs generated from expanded hiPSC-CMs displayed comparable contractility to those from unexpanded hiPSC-CMs, demonstrating uncompromised cellular functionality after expansion[9]. Nevertheless, even though the generation of EHTs from hiPSC-CMs has been well established, further developments are anticipated regarding the establishment of an HT assessment platform. Here, the rapid generation of large numbers of self-aggregating CS in 96-wells format allows an improvement in 3D conditions for HTS purposes. Overall, the advantage of a CSs as 3D cell culture is the option of using semiautomatic methods to produce spheroids such as pipetting robots for filling multi-well plates, exchanging medium, drug treatments, and finally analyzing the samples in high-content readers[20]. Here, we describe optimized protocols to generate high-purity and high-quality 3D CSs, which can be efficiently cryopreserved and screened for cardiac function by performing Ca2+ transient measurements using an optical calcium acquisition and analysis system. This model provides a simple, yet powerful, tool to perform high throughput screens on hundredsthousands of spheroids.[17,18]
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