Formulation and characterization of self-assembling cardiac spheroids as a model for drug-screening

Anticancer drug development needs to be shifted to a patient-specific platform that can be performed in high volume. Here, we report the generation and characterization of self-assembled cardiac spheroids formed from human induced pluripotent stem cells to be used for drug-screening applications. In doing so, we describe our employed cardiomyocyte differentiation and spheroid formation protocols to generate self-assembled spheroids whose sizes increased as a function of seeding density. We found that cardiac cells seeded at numbers of 2.5 x 10³, 10 x 10³, 15 x 10³, 20 x 10³ and 20 x 10³ resulted in the generation of spheroids with mean effective diameters of 264 ± 7 µm, 386 ± 4 µm, 445 ± 9 µm, 561 ± 10 µm, and 733 ± 8 µm, respectively. Using spheroid seeding with 10 x 10³ enabled the formation of spheroids with an approximate 386 ± 4 µm diameter, our desired size range emulative of the average in vivo intercapillary distance for downstream drug-treatment studies. We observed that implementing a centrifugation step during spheroid seeding greatly reduced any size variation without impacting the spheroid’s ability to form in-tact structures and retention of spontaneous beating properties. To quantify cardiomyocyte efficiency in our 2D differentiation protocols, we examined immunomarkers for cardiomyocyte and fibroblasts and observed an average cardiomyocyte prevalence of 4.86 ± 2.81 %, within our cell population. Lastly, we simulated acute drug-induced cardiotoxicity on the generated cardiac spheroids by examining their viability and spontaneous beating rate changes in response to doxorubicin-hydrochloride treatment. From the tested drug concentrations and our subsequent live fraction area curve fitting, we tabulated a half maximal inhibitory concentration of 2.11 µM, agreeable with reported concentrations in the clinic. Lastly, we observed that after 24 hours of drug exposure, statistically significant changes to the beating rates of spheroids exposed to concentrations ≥ 5 µM. Taken together, these data indicate that our stem cell-derived cardiac spheroids show promise as a potential patient-specific candidate model for assessing cardiotoxicity introduced by anticancer therapies.Mechanical Engineerin