356 Chapter 13 12). These cardiac spheroids were metabolically matured for several weeks. Here, we found that the spheroids cultured for 2 and 3 weeks were efficient in predicting the disturbed calcium handling in PLN-R14del hiPSC-CMs. Remarkably, however, 6-week-old spheroids showed a non-viable morphology and decreased calcium function. These findings suggest that the spheroids created with cells consisting of +80% hiPSC-CM, could represent a nonphysiological condition, rather than mimicking the cell types in the human myocardium. Recently, several studies have investigated the role of a controlled combination of hiPSCCMs, cardiac fibroblasts, and cardiac endothelial cells, which showed enhanced maturation and allowed high reproducibility across lines and differentiated cell batches of over a thousand microtissues. Interestingly, these three-cellular microtissues generated from an ACM patient, strikingly recapitulated features of the disease, indicating a multicell-type cause of genetic cardiomyopathies.37 Moreover, in another study, apoptotic cells became apparent in CM-only spheroids cultured for 8 weeks. Interestingly, spheroids generated from all four cardiac-cell types, in the ratio of CMs, ECs, SMCs, and CFs (4:2:1:1) remained viable throughout the culture period, by presumably following the distribution trends found in human myocardium.38 Where cardiac spheroids are simple 3D structures typically consisting of a cluster of cells, organoids are complex structures that aim to replicate the architecture and function of specific organs or tissues.39 Human heart organoids have been created since the mid-2010s, but only recently have significantly faithful models been achieved.40,41 The delay, when compared to other organoid types, is due to the specific obstacles posed by cardiac tissue. For example, nutrient distribution in organoids is a challenge, often resulting in a necrotic core and thereby limiting the maximum organoid size. Vascularization42 or longterm culture43 can promote the development and maturation of organoids, making them larger and more functional. The generation of human heart organoids by the self-assembly of hiPSCs could offer another possibility to mimic and culture human myocardium in vitro. Cardiac organoids generated by a three-step Wnt signaling modulation strategy mimic the age-matched human fetal cardiac tissues at the transcriptomic, structural, and cellular level44, cardiac proliferation45, and were able to model ischemic injury46. Subsequently, the improvement of cardiac spheroids and organoids by the combination of multiple cell types could improve physiological functions. However, this application on a very large scale, would remain challenging and very costly culture-wise. Moreover, mimicking the localization and interaction of the endocardial, myocardial, and epicardial layers in in vitro models would allow studying, for example, ventricular arrhythmias and stress-induced hypertrophy.47,48 Finally, the fusion of two subregional organoids, as shown with brain organoids49, could be a promising technique. For example, constructing specific-chambered hCOs with both ventricle-like and atrial-like structures or combining hCOs with vascular organoids50 may open the field of “next-generation” organoid technology in the near future. At this point, cardiac microtissues mimicking the 3D environment, yet remaining scalable and controllable51 offer a great opportunity for 3D disease modeling in a high-throughput screening (HTS) platform.
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