278 Chapter 11 SUMMARY Presented is a set of protocols for the generation and cryopreservation of cardiac spheroids (CSs) from human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) cultured in a high throughput multidimensional format. This three-dimensional (3D) model functions as a robust platform for disease modeling, high throughput screenings, and the screening of functional CSs after cryopreservation. ABSTRACT Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are of paramount importance for human cardiac disease modeling and therapeutics. We recently published a cost-effective strategy for the massive expansion of hiPSC-CMs in two dimensions (2D). Two major limitations are the cell immaturity and lack of three-dimensional (3D) arrangement and scalability in high-throughput-screening platforms. To overcome these limitations, the expanded cardiomyocytes form an ideal cell source for the generation of 3D cardiac cell culture and tissue engineering techniques. The latter holds great potential in the cardiovascular field, providing the next generation of more physiologically relevant highthroughput screens (HTS). Here, we present a scalable, HTS-compatible workflow for the generation, maintenance, and optical analysis of cardiac spheroids (CSs) in a 96-well-format. These small cardiac spheroids are essential to fill the gap present in current in vitro disease models and/or generation for 3D tissue engineering platforms. The resulting CSs possess a highly homogeneous morphology, size, and cellular composition. Furthermore, hiPSC-CMs cultured as CSs, display increased maturation. CSs present several functional features of the human heart such as spontaneous calcium handling and contractile activity. By automating the entire workflow from CSs generation to functional analysis, we enhance intra- and inter-batch reproducibility as demonstrated via high-throughput (HT) imaging and calcium handling analysis. The described protocol allows modeling of cardiac diseases and assessing drug/therapeutic effects at the single-cell level within a complex 3D cell environment in a fully automated HTS workflow. In addition, the study describes a straightforward procedure for whole-spheroids biobanking, thereby, providing researchers the opportunity to create next-generation living biobanks. HTS combined with long-term storage will substantially contribute to translational research in a wide range of areas, including drug discovery and testing, regenerative medicine, and the development of personalized therapies.
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