Abstract Body (Do not enter title and authors here): Human induced pluripotent stem cell (hiPSC)-derived engineered heart tissues (EHTs) have significant potential for advancing our understanding of cardiac physiology, modeling diseases, screening drugs, and developing cell therapies. A comprehensive evaluation of their functional maturation, intra- and intercell line variability, and pharmacological responsiveness is essential when developing safety or therapeutic applications.

We evaluated EHTs generated from three independent hiPSC-cardiomyocyte lines using the Mantarray contractility platform. Longitudinal tracking (day 2–day 42) captured morphological changes, contractile force, beat rate, and contraction-relaxation kinetics. We assessed intra- and interline variability of tissue development across multiple cell lines and differentiation batches. Starting day 4, tissues underwent continuous electrical stimulation, gradually increasing to 4 Hz by day 10, and were maintained at 4 Hz until day 28. Functional maturity was evaluated through the force-frequency relationship (FFR) test protocol as well as responses to external calcium, inotropes (Isoproterenol, Omecamtiv, Verapamil, and Blebbistatin), and cardiotoxic compounds (Digoxin, Doxorubicin, Sunitinib, and others).
Tissue widths reduced to approximately 40%, a sign of compaction observed by day 4 in all EHTs. Contractility stabilized by day 12, reaching 200 µN of contractile force and physiologically relevant spontaneous beating. Continuously paced tissues sustained a positive FFR (43% increase) up to 2.75 Hz compared to unstimulated tissue, exhibiting a 53% decrease in force when exposed to the FFR test protocol (n=8). All lines responded to positive and negative inotropes, external calcium at variable sensitivity. Contractile response to varied inotrope doses ranged from 2% to 8% CoV across multiple differentiation batches of the same cell line. Response pattern was consistent across cell lines, where variability in the onset and recovery timing of contractile function following cardiotoxic drug exposure was observed.

This study defines maturation timelines, functional performance benchmarks, and variability metrics for Mantarray EHTs across hiPSC lines. The ability to recapitulate physiologically relevant cardiac functions supports the readiness of these EHTs for preclinical applications. Genetic background and batch variability must be considered when designing cell-based therapeutic or safety pharmacology studies.