Despite the overwhelming use of cellularized therapeutics in cardiac regenerative engineering, approaches to biomanufacture engineered cardiac tissues (ECTs) at clinical scale remain limited. This study aims to evaluate the impact of critical biomanufacturing decisions—namely cell dose, hydrogel composition, and size-on ECT formation and function—through the lens of clinical translation. ECTs were fabricated by mixing human induced pluripotent stem-cell-derived cardiomyocytes (hiPSC-CMs) and human cardiac fibroblasts into a collagen hydrogel to engineer meso-(3 × 9 mm), macro- (8 × 12 mm), and mega-ECTs (65 × 75 mm). Meso-ECTs exhibited a hiPSC-CM dose-dependent response in structure and mechanics, with high-density ECTs displaying reduced elastic modulus, collagen organization, prestrain development, and active stress generation. Scaling up, cell-dense macro-ECTs were able to follow point stimulation pacing without arrhythmogenesis. Finally, we successfully fabricated a mega-ECT at clinical scale containing 1 billion hiPSC-CMs for implantation in a swine model of chronic myocardial ischemia to demonstrate the technical feasibility of biomanufacturing, surgical implantation, and engraftment. Through this iterative process, we define the impact of manufacturing variables on ECT formation and function as well as identify challenges that must still be overcome to successfully accelerate ECT clinical translation.

Myocardial infarction (MI) is a severe hypoxic event, resulting in the loss of up to one billion cardiomyocytes (CMs). Due to the limited intrinsic regenerative capacity of the heart, cell-based regenerative therapies, which feature the implantation of stem cell-derived cardiomyocytes (SC-CMs) into the infarcted myocardium, are being developed with the goal of restoring lost muscle mass, re-engineering cardiac contractility, and preventing the progression of MI into heart failure (HF). However, such cell-based therapies are challenged by their susceptibility to oxidative stress in the ischemic environment of the infarcted heart. To maximize the therapeutic benefits of cell-based approaches, a better understanding of the heart environment at the cellular, tissue, and organ level throughout MI is imperative. This review provides a comprehensive summary of the cardiac pathophysiology occurring during and after MI, as well as how these changes define the cardiac environment to which cell-based cardiac regenerative therapies are delivered. This understanding is then leveraged to frame how cell culture treatments may be employed to enhance SC-CMs’ hypoxia resistance. In this way, we synthesize both the complex experience of SC-CMs upon implantation and the engineering techniques that can be utilized to develop robust SC-CMs for the clinical translation of cell-based cardiac therapies.

Myocardial infarction is a leading cause of death worldwide and has severe consequences including irreversible damage to the myocardium, which can lead to heart failure. Cardiac tissue engineering aims to re-engineer the infarcted myocardium using tissues made from human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) to regenerate heart muscle and restore contractile function via an implantable epicardial patch. The current limitations of this technology include both biomanufacturing challenges in maintaining tissue integrity during implantation and biological challenges in inducing cell alignment, maturation, and coordinated electromechanical function, which, when overcome, may be able to prevent adverse cardiac remodeling through mechanical support in the injured heart to facilitate regeneration. Polymer scaffolds serve to mechanically reinforce both engineered and host tissues. Here, we introduce a novel biodegradable, customizable scaffold composed of wet-spun polycaprolactone (PCL) microfibers to strengthen engineered tissues and provide an anisotropic mechanical environment to promote engineered tissue formation. We developed a wet-spinning process to produce consistent fibers which are then collected on an automated mandrel that precisely controls the angle of intersection of fibers and their spacing to generate mechanically anisotropic scaffolds. Through optimization of the wet-spinning process, we tuned the fiber diameter to 339 ± 31 µm and 105 ± 9 µm and achieved a high degree of fidelity in the fiber structure within the scaffold (fiber angle within 1.8° of prediction). Through degradation and mechanical testing, we demonstrate the ability to maintain scaffold mechanical integrity as well as tune the mechanical environment of the scaffold through structure (Young’s modulus of 120.8 ± 1.90 MPa for 0° scaffolds, 60.34 ± 11.41 MPa for 30° scaffolds, 73.59 ± 3.167 MPa for 60° scaffolds, and 49.31 ± 6.90 MPa for 90° scaffolds), while observing decreased hysteresis in angled vs. parallel scaffolds. Further, we embedded the fibrous PCL scaffolds in a collagen hydrogel mixed with hiPSC-CMs to form engineered cardiac tissue with high cell survival, tissue compaction, and active contractility of the hiPSC-CMs. Through this work, we develop and optimize a versatile biomanufacturing process to generate customizable PCL fibrous scaffolds which can be readily utilized to guide engineered tissue formation and function.

Heart regeneration after myocardial infarction (MI) using human stem cell-derived cardiomyocytes (CMs) is rapidly accelerating with large animal and human clinical trials. However, vascularization methods to support the engraftment, survival, and development of implanted CMs in the ischemic environment of the infarcted heart remain a key and timely challenge. To this end, we developed a dual remuscularization-revascularization therapy that is evaluated in a rat model of ischemia-reperfusion MI. This study details the differentiation of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) for engineering cardiac tissue containing patterned engineered vessels 400 μm in diameter. Vascularized engineered human myocardial tissues (vEHMs) are cultured in static conditions or perfused in vitro prior to implantation and evaluated after two weeks. Immunohistochemical staining indicates improved engraftment of hiPSC-CMs in in vitro-perfused vEHMs with greater expression of SMA+ vessels and evidence of inosculation. Three-dimensional vascular reconstructions reveal less tortuous and larger intra-implant vessels, as well as an improved branching hierarchy in in vitro-perfused vEHMs relative to non-perfused controls. Exploratory RNA sequencing of explanted vEHMs supports the hypothesis that co-revascularization impacts hiPSC-CM development in vivo. Our approach provides a strong foundation to enhance vEHM integration, develop hierarchical vascular perfusion, and maximize hiPSC-CM engraftment for future regenerative therapy.

Acute myocardial infarction (MI) is a sudden, severe cardiac ischemic event that results in the death of up to one billion cardiomyocytes (CMs) and subsequent decrease in cardiac function. Engineered cardiac tissues (ECTs) are a promising approach to deliver the necessary mass of CMs to remuscularize the heart. However, the hypoxic environment of the heart post-MI presents a critical challenge for CM engraftment. Here, we present a high-throughput, systematic study targeting several physiological features of human induced pluripotent stem cell-derived CMs (hiPSC-CMs), including metabolism, Wnt signaling, substrate, heat shock, apoptosis, and mitochondrial stabilization, to assess their efficacy in promoting ischemia resistance in hiPSC-CMs. The results of 2D experiments identify hypoxia preconditioning (HPC) and metabolic conditioning as having a significant influence on hiPSC-CM function in normoxia and hypoxia. Within 3D engineered cardiac tissues (ECTs), metabolic conditioning with maturation media (MM), featuring high fatty acid and calcium concentration, results in a 1.5-fold increase in active stress generation as compared to RPMI/B27 control ECTs in normoxic conditions. Yet, this functional improvement is lost after hypoxia treatment. Interestingly, HPC can partially rescue the function of MM-treated ECTs after hypoxia. Our systematic and iterative approach provides a strong foundation for assessing and leveraging in vitro culture conditions to enhance the hypoxia resistance, and thus the successful clinical translation, of hiPSC-CMs in cardiac regenerative therapies.