358 Chapter 13 cardiotoxicity maybe even more than 16 hiPSC lines need to be considered. The variability appears to be less relevant for hiPSC-CM disease modeling and drug screening but still, studies with more than one line should be considered. Overall, the recapitulation of the prediction in hiPSC-CM models could be impeded by the baseline differences in the hiPSC cultures (e.g., hiPSC line ancestry, HLA haplotype, differentiation efficiency, hiPSC-CM maturity level, and epigenetics). Here, the individual genetic variation in ion channels, sarcomeric protein expression, and drug response in healthy control hiPSC-CM supports the request for isogenic controls or multiple hiPSC clones and the standardization and automatization of many patientderived hiPSC-CMs in our scientific community. High-throughput drug screening of hiPSC-CMs models As discussed, studies using more than 4 hiPSC lines for cardiac research in vitro are rare, yet elaborating the molecular mechanism of complex CM features requires many more hiPSC lines in future in vitro hiPSC-CM models. A predictive, quantitative, and reproducible highthroughput screening (HTS) pipeline is required to design, mimic the clinical issues, and guide the development of phenotypic-specific therapeutics. For phenotypic HTS fixed-endpoint assays such as the identification and localization of proteins of interest can be used to evaluate the static properties of cardiac cells. In this thesis, we observed significant increases in lipid droplets due to metabolic disruption (Chapter 8), followed by the activation of XBP-1, indicative of activated UPR (Chapter 9), and the increased fibroblast activation (Chapter 12) in hiPSC-CMs of patients with PLN-R14del mutation.These findings provided valuable information about the cardiac pathophysiology and the molecular mechanism, such as the ability to ameliorate the phenotype in hiPSC-CM models. As previously described in this thesis, 2D models have proven to be less effective for the prediction of drug efficacy and disease modeling. Here our published framework from Chapter 4 (expanded hiPSC-CMs) and Chapter 11 (cardiac spheroids) in a 384 wells HTS format could provide an improved hiPSC-CM model, for the initial screening of morphological, cell death, gene, protein, and functional assessments. To screen complex physiological phenomena such as CM function, a more complex analysis with optical Ca2+ or voltage sensors, action potential, or force can be performed. Previously, the multi-/micro-electrode array (MEA) system has provided a non-invasive user-friendly platform consisting of dot-like electrodes measuring fluctuations in the extracellular field potential of a complete monolayer for detailed electrophysiological analysis. Although both MEA and macro-tissue platforms have the advantage of plug-and-play commercial read-out platforms, both have some throughputlimiting disadvantages of expensive and high-cell number plate formats, analogous levels of process-induced and batch-to-batch variations, as the CM-specific event could be obscured by averaging the complete field of view of a culture well.
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