Biomanufacturing Vascularized Cardiac Tissue at Therapeutic Scale

Abstract

Engineering thick, viable cardiac tissue for regenerative medicine requires the incorporation of perfusable vasculature that can sustain metabolically active cells throughout the construct. Every cardiomyocyte in the human heart sits within 100 µm of the nearest blood vessel, yet engineered tissues lack the hierarchical vascular networks needed to deliver oxygen and nutrients beyond the diffusion limit. Without functional vasculature, engineered cardiac tissues are restricted to thicknesses below ~200 µm, fundamentally limiting their therapeutic use. The ability to embed immediately perfusable, hierarchical vascular networks within engineered cardiac constructs would provide more physiologically relevant constructs for drug screening, disease modeling, and regenerative therapies. My Ph.D. thesis led to two critical and complementary advances in engineering vascularized cardiac tissues. The first advance is an integrated experimental and computational platform for quantifying and predicting oxygen transport within perfused three-dimensional tissue constructs. Central to this effort is the use of a genetically encoded fluorescent hypoxia reporter cell line (HEK-UnaG) that enables spatial mapping of oxygen concentration across entire tissue cross-sections, coupled with a custom 3D-printed perfusion system that provides independent control over vessel diameter, tissue dimensions, and flow rate. To further assess oxygen diffusion and uptake, we built a computational model of oxygen transport parameterized by experimentally iii measured data, which is validated by two independent parametric sweeps – varying perfusion rate and vessel diameter. Importantly, we find a mean error of 3.6% between model predictions and experimental measurements across all conditions tested. Using this validated computational platform, we demonstrated that smaller diameter vessels yield the most efficient oxygenation of densely cellular tissues. By introducing an immediately perfusable network of mesoscale vessels (D = 100 µm) formed from sacrificial gelatin fibers incorporated above their percolation threshold (Φ ~ 0.1) we could significantly increase the maximum tissue viability zone. In densely cellular constructs (50×106 cells/mL, macrovessel spacing = 2 mm) perfused solely through their macrovessels, the maximum tissue viability zone is ~240 µm from the nearest macrovessel. By contrast, tissue constructs that contained perfusable macrovessels and mesovessels, generated by the removal of sacrificial gelatin fibers (Φ = 0.35) embedded within the tissue, exhibited high cell viability across the full tissue width (i.e., 2 mm viability zone). The second advance is the development of a novel biofabrication method known as, coaxial sacrificial writing into functional tissue (co-SWIFT). co-SWIFT enables one to directly embed hierarchically branching, biomimetic vascular networks within both acellular and densely cellular tissue matrices. We designed a novel coaxial printhead with an extended core-shell nozzle that allows both core-core and shell-shell connections to be immediately established between printed vessels, unlocking the ability to create branching vascular architectures. Next, we determined the requisite rheological properties for co-SWIFT printing and demonstrated the fabrication of multigenerational branching networks that obey Murray’s law within (1) transparent alginate, (2) microporogen-structured collagen, and (3) densely cellular cardiac organ building block (OBB) matrices. Upon removal of the sacrificial core, the resulting lumens are wrapped by smooth muscle cells and subsequently seeded with endothelial cells to produce a confluent endothelium that iv provides good barrier function. Cardiac co-SWIFT tissues contract synchronously within days of fabrication and exhibit cardio-effective drug responses. In summary, my PhD thesis establishes both a quantitative framework to guide the rational design of perfusable vascular networks in engineered tissues and a generalizable fabrication method for embedding biomimetic vasculature within organ-specific constructs. Together, these advances address two critical unmet needs in the field: (1) understanding how vascular architecture governs oxygen transport and cell viability in engineered tissues, and (2) embedding multi-layered, branching vasculature within cellularly dense tissues. Looking ahead, we envision combining these advances to generate thick, vascularized cardiac tissues with physiologically relevant cell density, architecture, and function for regenerative medicine applications.