José Manuel Rivera-Arbelaez

Traditional two-dimensional cell cultures and in vivo animal studies fail to fully recapitulate human cardiac physiology, highlighting the urgent need for more relevant human-based models. Engineered three-dimensional cardiac systems - including organoids, engineered heart tissues, and heart-on-chip platforms offer promising alternatives, providing structural and functional insights into cardiac biology. However, a critical limitation of these models is their inability to perform fluid pumping and relaxation, which together define fundamental heart function. Engineered cardiac chambers have emerged to address this gap, enabling physiologically relevant pressure-volume measurements and capturing both contractile and diastolic dynamics that mimic aspects of native cardiac hemodynamics. This mini-review examines the current state of engineered cardiac chambers and highlights their main design features. We discuss their applications in disease modeling and drug testing, and outline key factors influencing the optimization of these models, including balancing biological fidelity with process efficiency through modular design principles. Overall, engineered cardiac chambers represent a unique, powerful platform to improve mechanistic understanding of cardiac disease, offering significant potential to advance cardiovascular research and therapeutic development.

The maturation of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) remains a major challenge in developing functional in vitro cardiac models. While three-dimensional (3D) culture systems improve structural and metabolic properties, they do not fully recapitulate adult cardiomyocyte physiology. Exogenous electrical stimulation has emerged as complementary strategy to further drive maturation. This review highlights both the maturation effects of electrical pacing in 3D cardiac tissues, including enhanced sarcomere organization, conduction velocity, calcium handling, and contractile function. It also discusses the technological parameters used to achieve these outcomes, such as electric field voltage (EFV), pulse duration (PD), stimulation waveform, electrode materials, and pacing protocols, and how these factors influence hPSC-CM development. Despite progress, further research is needed to optimize stimulation setups and to integrate electrical pacing with other maturation cues. Advancing high-throughput, miniaturized platforms will be essential for translating these models into biomedical applications like drug discovery and disease modeling.

Engineered heart tissues (EHTs) have shown great potential in recapitulating tissue organization, functions, and cell-cell interactions of the human heart in vitro. Currently, multiple EHT platforms are used by both industry and academia for different applications, such as drug discovery, disease modelling, and fundamental research. The tissues’ contractile force, one of the main hallmarks of tissue function and maturation level of cardiomyocytes, can be read out from EHT platforms by optically tracking the movement of elastic pillars induced by the contractile tissues. However, existing optical tracking algorithms which focus on calculating the contractile force are customized and platform-specific, often not available to the broad research community, and thus hamper head-to-head comparison of the model output. Therefore, there is the need for robust, standardized and platform-independent software for tissues’ force assessment. To meet this need, we developed ForceTracker: a standalone and computationally efficient software for analyzing contractile properties of tissues in different EHT platforms. The software uses a shape-detection algorithm to single out and track the movement of pillars’ tips for the most common shapes of EHT platforms. In this way, we can obtain information about tissues’ contractile performance. ForceTracker is coded in Python and uses a multi-threading approach for time-efficient analysis of large data sets in multiple formats. The software efficiency to analyze circular and rectangular pillar shapes is successfully tested by analyzing different format videos from two EHT platforms, developed by different research groups. We demonstrate robust and reproducible performance of the software in the analysis of tissues over time and in various conditions. ForceTracker’s detection and tracking shows low sensitivity to common incidental defects, such as alteration of tissue shape or air bubbles. Detection accuracy is determined via comparison with manual measurements using the software ImageJ. We developed ForceTracker as a tool for standardized analysis of contractile performance in EHT platforms to facilitate research on disease modeling and drug discovery in academia and industry.

The synchronization of the electrical and mechanical coupling assures the physiological pump function of the heart, but life-threatening pathologies may jeopardize this equilibrium. Recently, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have emerged as a model for personalized investigation because they can recapitulate human diseased traits, such as compromised electrical capacity or mechanical circuit disruption. This research avails the model of hiPSC-CMs and showcases innovative techniques to study the electrical and mechanical properties as well as their modulation due to inherited cardiomyopathies. In this work, hiPSC-CMs carrying either Brugada syndrome (BRU) or dilated cardiomyopathy (DCM), were organized in a bilayer configuration to first validate the experimental methods and second mimic the physiological environment. High-density CMOS-based microelectrode arrays (HD-MEA) have been employed to study the electrical activity. Furthermore, mechanical function was investigated via quantitative video-based evaluation, upon stimulation with a β-adrenergic agonist. This study introduces two experimental methods. First, high-throughput mechanical measurements in the hiPSC-CM layers (xy-inspection) are obtained using both a recently developed optical tracker (OPT) and confocal reference-free traction force microscopy (cTFM) aimed to quantify cardiac kinematics. Second, atomic force microscopy (AFM) with FluidFM probes, combined with the xy-inspection methods, supplemented a three-dimensional understanding of cell-cell mechanical coupling (xyz-inspection). This particular combination represents a multi-technique approach to detecting electrical and mechanical latency among the cell layers, examining differences and possible implications following inherited cardiomyopathies. It can not only detect disease characteristics in the proposed in vitro model but also quantitatively assess its response to drugs, thereby demonstrating its feasibility as a scalable tool for clinical and pharmacological studies.

Advanced in vitro models that recapitulate the structural organization and function of the human heart are highly needed for accurate disease modeling, more predictable drug screening, and safety pharmacology. Conventional 3D Engineered Heart Tissues (EHTs) lack heterotypic cell complexity and culture under flow, whereas microfluidic Heart‐on‐Chip (HoC) models in general lack the 3D configuration and accurate contractile readouts. In this study, an innovative and user‐friendly HoC model is developed to overcome these limitations, by culturing human pluripotent stem cell (hPSC)‐derived cardiomyocytes (CMs), endothelial (ECs)‐ and smooth muscle cells (SMCs), together with human cardiac fibroblasts (FBs), underflow, leading to self‐organized miniaturized micro‐EHTs (µEHTs) with a CM‐EC interface reminiscent of the physiological capillary lining. µEHTs cultured under flow display enhanced contractile performance and conduction velocity. In addition, the presence of the EC layer altered drug responses in µEHT contraction. This observation suggests a potential barrier‐like function of ECs, which may affect the availability of drugs to the CMs. These cardiac models with increased physiological complexity, will pave the way to screen for therapeutic targets and predict drug efficacy.

The high rate of drug withdrawal from the market due to cardiovascular toxicity or lack of efficacy, the economic burden, and extremely long time before a compound reaches the market, have increased the relevance of human in vitro models like human (patient‐derived) pluripotent stem cell (hPSC)‐derived engineered heart tissues (EHTs) for the evaluation of the efficacy and toxicity of compounds at the early phase in the drug development pipeline. Consequently, the EHT contractile properties are highly relevant parameters for the analysis of cardiotoxicity, disease phenotype, and longitudinal measurements of cardiac function over time. In this study, we developed and validated the software HAARTA (Highly Accurate, Automatic and Robust Tracking Algorithm), which automatically analyzes contractile properties of EHTs by segmenting and tracking brightfield videos, using deep learning and template matching with sub‐pixel precision. We demonstrate the robustness, accuracy, and computational efficiency of the software by comparing it to the state‐of‐the‐art method (MUSCLEMOTION), and by testing it with a data set of EHTs from three different hPSC lines. HAARTA will facilitate standardized analysis of contractile properties of EHTs, which will be beneficial for in vitro drug screening and longitudinal measurements of cardiac function.

The use of Engineered Heart Tissues (EHT) as in vitro model for disease modeling and drug screening has increased, as they provide important insight into the genetic mechanisms, cardiac toxicity or drug responses. Consequently, this has highlighted the need for a standardized, unbiased, robust and automatic way to analyze hallmark physiological features of EHTs. In this study we described and validated a standalone application to analyze physiological features of EHTs in an automatic, robust, and unbiased way, using low computational time. The standalone application “EHT Analysis” contains two analysis modes (automatic and manual) to analyzes the contractile properties and the contraction kinetics of EHTs from high speed bright field videos. As output data, the graphs of displacement, contraction force and contraction kinetics per file will be generated together with the raw data. Additionally, it also generates a summary file containing all the data from the analyzed files, which facilitates and speeds up the post analysis. From our study we highlight the importance of analyzing the axial stress which is the force per surface area (μN/mm²). This allows to have a readout overtime of tissue compaction, axial stress and leave the option to calculate at the end point of an experiment the physiological cross-section area (PSCA). We demonstrated the utility of this tool by analyzing contractile properties and compaction over time of EHTs made out of a double reporter human pluripotent stem cell (hPSC) line (NKX2.5^EGFP/+-COUP-TFII^mCherry/+) and different ratios of human adult cardiac fibroblasts (HCF). Our standalone application “EHT Analysis” can be applied for different studies where the physiological features of EHTs needs to be analyzed under the effect of a drug compound or in a disease model.

Cardiomyocytes derived from human pluripotent stem cells (hPSC-CMs) hold a great potential as human in vitro models for studying heart disease and for drug safety screening. Nevertheless, their associated immaturity relative to the adult myocardium limits their utility in cardiac research. In this study, we describe the development of a platform for generating three-dimensional engineered heart tissues (EHTs) from hPSC-CMs for the measurement of force while under mechanical and electrical stimulation. The modular and versatile EHT platform presented here allows for the formation of three tissues per well in a 12-well plate format, resulting in 36 tissues per plate. We compared the functional performance of EHTs and their histology in three different media and demonstrated that tissues cultured and maintained in maturation medium, containing triiodothyronine (T3), dexamethasone, and insulin-like growth factor-1 (TDI), resulted in a higher force of contraction, sarcomeric organization and alignment, and a higher and lower inotropic response to isoproterenol and nifedipine, respectively. Moreover, in this study, we highlight the importance of integrating a serum-free maturation medium in the EHT platform, making it a suitable tool for cardiovascular research, disease modeling, and preclinical drug testing.