Scalable Production of Uniform Cardiac Tissues Utilizing Hydrogel-Supported Microsphere Tissues with Applications to Large Cardiac Tissue Production

Abstract

To meet the high demand of cardiomyocytes for use in regenerative medicine, cardiotoxicity studies, and disease modeling, this work set out to produce engineered cardiac microtissues scalably with high uniformity post differentiation in terms of their size and shape, their cell type composition, and their resulting functionality. Moreover, this work set out to demonstrate the application of microtissues for use in large tissue production. To achieve this, in project 1 (Chapter 2), scaffold-free cardiac aggregates were compared to hydrogel-based cardiac microspheres. Hydrogel-based microspheres showed uniform, functional tissue production and it was shown that hiPSCs differentiated in 3D produce a range of cardiac cell types important for engineered tissue function including cardiomyocytes, endocardial cells, endothelial cells, fibroblasts, mesenchymal cells, and smooth muscle cells. In project 2 (Chapter 3), aggregates and microspheres were further assessed using bioinformatic analyses. Notably, it was also observed that when microsphere cardiomyocytes were directly compared to aggregate cardiomyocytes, that microsphere cardiomyocytes shift to fatty acid metabolism, driving the initial observed fatty acid metabolism observed in pseudobulk analyses in Chapter 2. Together, these results helped better our understanding of cell-type specific roles driving observed tissue differences in Chapter 2. In project 3 (Chapter 4), microspheres were selected to investigate daily changes in cell types, cell signaling, and tissue ECM and stiffness. HiPSC cardiomyocyte development was modeled and a database of healthy 3D cardiac differentiation in hydrogel-supported microspheres was produced for use in comparing to diseased hiPSC cardiac differentiations. Additionally, tissue stiffness and collagen deposition increased throughout differentiation while extracellular matrix protein expression both increased and decreased throughout differentiation. In project 4 (Chapter 5), microsphere starvation protocols were investigated to improve cardiac tissue cell type compositions. It was found that to improve cell type composition and function in engineered cardiac microspheres, treatment of no glucose media to select cardiac cell types followed by maturation media treatment for functionality is beneficial. Lastly, in project 5 (Chapter 6), cardiac microsphere microtissues were utilized for large engineered cardiac tissues assembly. Tissues assembled on day 5 showed faster contraction velocities later in culture and were therefore used to assemble a perfused cm-scale tissue within a large tissue bioreactor. Together, this work provides a toolset for producing large engineered cardiac tissues using a simple, one-step assembly of building block tissues. Together, innovative engineering techniques were developed to produce cardiac tissues with greater uniformity and function. Moreover, diffusion challenges were overcome in cm-scale tissues utilizing hydrogel-supported microspheres, working towards scalable biomanufacturing a high number of cardiomyocytes.