One Billion hiPSC-Cardiomyocytes: Upscaling Engineered Cardiac Tissues to Create High Cell Density Therapies for Clinical Translation in Heart Regeneration

Abstract

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.

Advances in cardiac tissue engineering and heart‐on‐a‐chip

“…Scalable tissue production requires generating enough cardiomyocytes form human iPSC and ESC, as billions of cells are required for the generation of large patches for transplantation. 201,203 Recent studies describe protocols that allow for efficient cardiomyocyte expansion. 204 Founded on these advances, the approaches from machine learning and artificial intelligence may further allow for the development of more effective approaches that would drive combinatorial screening and discovery of molecule combinations to achieve a large-scale expansion of cardiomyocytes.…”

Patterned Arteriole-Scale Vessels Enhance Engraftment, Perfusion, and Vessel Branching Hierarchy of Engineered Human Myocardium for Heart Regeneration

“…First, differentiated hiPSC-CMs exhibit an immature, fetal-like phenotype [ 22 , 23 , 24 ]. While many groups have used mechanical [ 25 , 26 ], electrical [ 27 ], or biomolecular [ 28 , 29 , 30 , 31 ] methods to mature the contractile apparatus, gene expression, electrophysiology, and metabolism of hiPSC-CMs, the appropriate level of maturity for transplantation in vivo that maximizes survival (e.g., in relative ischemia) and contractile function has not been established. Recent literature suggests that immature hiPSC-CMs may engraft better onto the injured heart [ 32 ], which is thought to be due to their relatively immature metabolic profile [ 33 ], although in vitro-matured hiPSC-CMs that survive implantation appear to have more adult-like structural features [ 24 , 34 ].…”

“…Empirical evaluation of co-culture conditions is sparse in the tissue engineering literature, yet it is of broad concern and warrants study. Finally, the throughput and ease of scalable biomanufacturing are slowed by increasingly complex tissue and vascular geometries [ 4 , 37 ] and challenged by the fundamental difficulties in handling hiPSC-CMs and requiring high CM density for syncytium formation [ 31 , 38 , 39 ]. These considerations have influenced the design of our vascularized engineered human myocardial tissues (vEHMs), from biomaterial selection and vessel patterns to methods of endothelialization, culture, and implantation.…”