291 Generation, high throughput screening, and biobanking of human iPSC-derived cardiac spheroids 11 DISCUSSION Cardiac drug discovery is hampered by the reliance on non-human animal and cellular models with inadequate throughput, and physiological fidelity, to accurately perform readouts. The hiPSC-cardiomyocyte biology coupled with HT instrumentation and physiological probes has the potential to re-introduce human models into the earliest stages of cardiac disease modeling and drug discovery. We developed a 3D cardiac tissue generation method that produces high-quality and functional CSs for an optimal cardiac disease modeling and drug screening platform. Our method relies on several crucial factors and is a variant of existing protocols[9,10,28]. These methods include; 1) generation of 3D tissue constructs, 2) the optimal cell number and timing before the screening, 3) improving sensitivity and high throughput capacity of instruments, and 4) being able to freeze the spheroids before any functional analysis. Unlike the previously described protocols, the proposed protocol describes the generation of up to 1500 spheroids per day and the suitability for HTS. Conventional analysis of a hundred compounds over 6 x ½ log doses for 10 replicates using existing 96-well calcium imaging systems or 24-well multiplexed engineered heart tissues would require approximately 500 million to 3 billion hiPSC-CMs[31]. The proposed application makes cardiac screenings less costly and time effective compared to the conventional systems since the 96 wells plates required only 10% of the seeding density compared to the described method. This small 3D model mimics the biological and physiological phenotype of the in vivo cardiovascular environment. As previously demonstrated, calcium transients dramatically increase in 3D cardiac tissue constructs as compared to 2D monolayer cell cultures[32]. Next, we found that the seeding density and proper culturing time are also critical factors for a successful CS screening. The densities of 10K-20K hiPSC-CMs per spheroid and the screening between week 2-3 after generation seemed optimal, whereas too small or too old spheroids show disturbed calcium handling (Figure 2 and Figure 3). Therefore, it is of importance to maintain seeding densities as consistently as possible, since size influences the functional parameters. Also, although this optical method provides good results for live 3D cultures as a whole tissue, obtaining data within larger spheroids at (sub-)cellular level is challenging without relying on time-consuming histology methods. Recently, several approaches have been published that used “optically clearing”, which enables the acquisition of whole 3D organoids with the opportunity for single-cell quantification of markers. Here, we adapted a three-day protocol from hCS harvesting to image analysis, which is optimized for 3D imaging using confocal microscopy[29](Figure 1C and Figure 4D). Lastly, with the increase in 3D cardiac tissue applications and commercial applications, the demand for long-term storage and patient-specific biobanking from various donors is rising. Cryopreservation is an effective strategy to generate HTS-plates from multiple batches over time. The freezing of hiPSC-CMs has been described previously and is not different compared to other cultured cell types[10,33,34]. Recently, approaches for freezing plates with 2D cells have
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