Compound Screening using Cardioid technology

1. Modeling doxorubicin-induced cardiotoxicity and cardioprotection for high content screening using Cardioids 

To validate the utility of Cardioids as a robust platform for assay development and high-throughput screening, we tested their ability to model known drug-induced cardiotoxicity using the clinically relevant chemotherapeutic agent doxorubicin. This proof-of-concept study demonstrates both the sensitivity of Cardioids to a cardiotoxic compound and their applicability for evaluating cardioprotective strategies. 

Doxorubicin is a widely used chemotherapeutic agent known for its cardiotoxic side effects. To evaluate the potential of Cardioids in modeling cardioprotective strategies, we co-administered a cardioprotective compound alongside doxorubicin. Treatment began on Day 21, with three sequential doses administered every 48 hours via media exchange. Functional responses were tracked using specifically developed calcium transient-based assays to assess both the impact of doxorubicin and the protective effects of the cardioprotectant. 

Across multiple batches, Cardioids consistently exhibited a clear toxic phenotype at an effective doxorubicin concentration of 0.68 µM, closely mirroring concentrations reported in physiological studies. This consistency underscores the reliability and accuracy of the Cardioid model in recapitulating the cardiotoxic response. Notably, Cardioids co-treated with the cardioprotectant showed significant mitigation of toxicity, demonstrating the system’s utility not only in detecting harmful effects but also in evaluating compounds that can prevent or reverse cardiac damage. 

This study underscores the strength of Cardioids as a physiologically relevant model for both cardiotoxicity detection and therapeutic intervention testing. By faithfully replicating doxorubicin-induced toxicity and demonstrating protection through co-treatment, Cardioids are a powerful system for both preclinical safety screening and drug discovery workflows.

‍Figure 1: Left – Representative images of mid-section of Cardioids under three conditions: untreated (healthy), treated with doxorubicin, and co-treated with doxorubicin and a cardioprotective compound. Cardioids were fixed and stained using an anti-TNNT2 antibody. Right – Quantification of cardiomyocyte toxicity based on TNNT2 staining.

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2. Modeling Hypertrophic Cardiomyopathy (HCM) in Cardioids  

To assess the potential of Cardioids in disease modeling and phenotypic screening, we established an assay in Cardioids to model drug-induced hypertrophic cardiomyopathy (HCM) by inducing hypercontractility. We then proceeded to utilize this model and assay to show our platforms’ ability to perform automated high-throughput screening campaigns. 

Human iPSC-derived Cardioids were cultured in 384-well plates. Cardioids were quality controlled for morphological and functional parameters that were assessed throughout development to ensure consistency and robustness. The treatment regimen began with the first administration of the HCM-inducing drug, co-delivered with the compound of interest, on Day 21. A second administration was performed on Day 23, and Cardioids were subsequently imaged and functionally analyzed on Day 25 to assess the hypertrophic response. 

Chronic exposure to the drug led to a significant increase in cardiomyocyte size, consistent with the hypertrophic remodeling seen in disease states. This enlargement was however accompanied by a reduction in Cardioid cavity size, resulting in an overall decrease in total Cardioid area (hypertrophy at the expense of the inner chamber cavity). We then proceeded to score this decrease as a phenotypic proxy for quantifying hypertrophy. As a control, treatment with Mavacamten (red dot in the plot) — a clinically validated myosin-inhibitor — effectively countered the drug-induced changes, preserving Cardioid structure and preventing chamber decrease and concomitant area loss. 

Each compound was screened in both biological and technical replicates, with at least eight Cardioids evaluated per condition. From this screening, we identified 73 total hits. These comprise of positive hits that reversed or mitigated the drug-induced hypertrophic phenotype (left end of the plot) as well as toxic compounds which are evident on the right end of plot, marked by a substantial reduction in Cardioid size and complete loss of calcium transients. 

This HCM screen highlights the potential of Cardioids and the HBB platform as a powerful tool for modeling disease phenotypes and screening therapeutic interventions. The system accurately captures structural and functional changes associated with drug-induced hypertrophy, while also enabling detection of both protective and toxic compound responses in a high-throughput format.

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‍Figure 1: Left – Results from a 1,280-compound pilot screen showing normalized area changes in Cardioids relative to the negative control (HCM inducing drug-treated only). The positive control, representing co-treatment with Mavacamten, demonstrates a clear prevention of the hypertrophic phenotype. Right – Representative images from the screen highlighting phenotypic differences across treatment conditions.