Data-Driven Model Development for Cost-Effective, Reliable Cardiac Tissue Manufacturing

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U.S. mortality rates from heart disease are increasing, driven particularly by deaths in younger adults. Limited availability of native human heart tissue impedes research, drug discovery, and clinical cardiac regeneration efforts. Treatment with stem cell-derived heart tissues, composed of functional contracting heart cells called cardiomyocytes, has high potential to achieve clinically meaningful outcomes. However, the number of cells required for therapeutic benefit has been estimated to be 1-10 billion per patient. Technical challenges and limited scalability of current processes for manufacturing engineered heart tissues hinder progress toward clinical use. The primary goal of this research project is to design reliable and cost-effective manufacturing processes for producing engineered heart tissues. The project employs a novel scalable, one-step heart tissue production platform, combines experimental and modeling studies, and leverages their integration to understand how different operating conditions impact the heart tissue manufacturing process. The research has the potential to transform the ability to manufacture engineered heart tissue for drug testing and heart regeneration applications. Workforce development aspects of the project include engagement of undergraduate and graduate students and practicing engineers through joint upper level curriculum development, teaming with ongoing efforts to support local industry, including establishing a biomanufacturing track for the new cross-disciplinary Advanced Manufacturing engineering specialization, and collaborating with Alabama State University's Life Sciences Department to train students in fundamentals important to biomanufacturing, tissue engineering and bioprinting. To support long-term workforce development, this interdisciplinary research is being incorporated into multiple K-12 outreach efforts. Undergraduate researchers and Alabama State University summer interns will be key members of the research team. Technical challenges and limited scalability of current processes for manufacturing engineered cardiac tissues, including the ubiquitous use of pre-differentiated cardiomyocytes (CMs), has hindered progress toward clinical use. Lack of CM maturation and variable outcomes in scalable stirred flask bioreactors are critical barriers in therapeutic CM production. Professor Lipke has recently established a novel platform to directly differentiate hydrogel-encapsulated human induced pluripotent stem cells (hiPSCs) into engineered cardiac tissues. The research employs a custom microfluidic system to rapidly encapsulate hiPSCs in highly uniform microspheroids with controllable size and shape. By modifying tissue axial ratio, differentiating CMs will experience anisotropic mechanical stimulation during spontaneous contraction; this has been typically impossible to achieve in scalable, suspension cultures and has the unique potential to drive CM maturation. To leverage this potentially transformative approach for single-stage cardiac tissue production, a robust manufacturing process is needed. Thus, the overarching goal of this project is to design reliable and cost-effective manufacturing processes for reproducible, cost-effective, and high-quality production of cardiac tissues. To achieve this goal, the project pursues 3 aims: (1) Investigate the characteristics of engineered cardiac tissues formed using a single-stage process of human induced pluripotent stem cell (hiPSC) hydrogel microspheroid encapsulation and direct differentiation in suspension culture, (2) Identify regions of optimal or near-optimal parameter ranges to manufacture engineered cardiac tissues reliably in suspension culture and spinner flask bioreactor systems using data-driven models, and (3) Test the ability of hydrogel microspheres to support single-stage engineered cardiac tissue manufacturing in a more readily scalable, spinner flask bioreactor system. Guided by data-driven models, the project investigates the effects of hydrogel microspheroid shape and size, polymer precursor concentration, crosslinking time and light intensity, and applied shear stress, on safety and efficacy attributes of single-stage manufactured cardiac tissues. Real-time functional monitoring and in-depth initial and end-point characterization are being performed to assess initial and resulting cell phenotypes. The approach integrates experimental studies and data-driven models through design of adaptive experimental campaigns for manufacturing cardiac tissue in a single-stage process directly from hydrogel encapsulated hiPSCs. Using inverse analysis on the data-driven models, the range of process parameters is being determined to minimize variability in electrophysiological and mechanical contraction attributes of engineered cardiac tissues, which is essential for their reliable manufacturing. Fundamentals of tissue engineering, polymer science, and microfluidics will be drawn upon to manufacture cardiac tissue microspheres directly from hiPSCs, rather than employing pre-differentiated cardiac cells. This approach is unique and, by eliminating multiple cell handling steps and employing a polyethyleneglycol-fibrinogen photocrosslinkable matrix, it has the potential to provide a transformative advance in the ability to manufacture engineered heart tissue for drug testing and cardiac regeneration applications.