PROJECT SUMMARY The convergence of human induced pluripotent stem cells (hiPSCs), organoids, synthetic biology and 3D bioprinting promises a future of patient-specific lab-grown organs for patients suffering from organ failure. However, to realize this organ engineering vision, biofabrication researchers sorely need thousand-liter-scale cultures of hiPSCs to generate enough material to begin high-throughput experimentation. Solving the myriad challenges in organ construction, vascularization, maintenance, maturation, and characterization will require decades of painstaking research. Yet, deriving patient-specific cells at this scale remains two orders of magnitude too expensive for academic laboratories due, in large part, to the expensive growth factors required for hiPSC maintenance and differentiation. Furthermore, existing protocols to generate organoids from stem cells are cumbersome, slow, and inefficient, limiting the number of organoids that can be derived for 3D bioprinting applications. In these proposed studies, we detail novel methods to dramatically reduce the cost of stem cell maintenance and increase the scale of organoid production. To reduce the costs of large-scale hiPSC growth by two orders of magnitude, we propose to engineer growth factor-free hiPSCs by programming them to express constitutively-active growth factor receptors which can be excised prior to differentiation. To enhance the scale and throughput in generating multicellular cardiac organoids, we propose engineering hiPSCs to undergo simultaneous multicellular differentiation without requiring growth factors. To achieve this, we propose a novel stochastic Cre-lox recombination system to upregulate one-of-three transcription factors, EOMES, Nkx3.1, or ETV2, to generate tri-cellular synthetic cardiac organoids containing cardiomyocytes, fibroblasts, and endothelial cells, respectively. By culturing millions of these synthetic cardiac organoids in suspension culture, we will derive therapeutically-relevant quantities of densely cellular myocardial bioink for 3D bioprinting. We will next use synthetic cardiac organoid bioink to derive a human-scale, thick-walled, and vascularized ventricle model. These bioprinted ventricles will be housed in a custom perfusion bioreactor for studying how mechanical and electrical stimulation can maintain vascular perfusion, enhance cardiomyocyte maturation and alignment, and affect organ- scale contractility and ejection fraction. The highly scalable stem cell and organoid culture methods presented here are applicable across many organ systems, and could revolutionize the scale and pace of organ biofabrication research.

PROJECT NARRATIVE Progress towards the goal of bioprinting organs for therapeutic purposes has been greatly slowed by our inability to affordably grow, and efficiently differentiate, the large numbers of stem cells required. Here, we propose novel methods to dramatically reduce the costs of large-scale cultures and to co-differentiate multiple cell types in a single bioreactor to form synthetic cardiac organoids for 3D bioprinting human-scale ventricle models. These advances will significantly accelerate our ability to perform upcoming decades of research and translational development required to reach the goal of on-demand autologous organs for transplantation.