Programming Complex Alignment in Engineered Cardiac Tissue

Abstract

By replicating the structure and function of the human heart, cardiac tissue engineers aim to transform our understanding of the biomechanical drivers of heart disease and enable the development of novel drugs and curative therapies. Seminal advances in cardiac tissue engineering reveal that replicating key physiological attributes of the human myocardium can unlock disease mechanisms that were previously unidentifiable.1,2 Similarly, advanced tissue engineered cardiac tissue constructs are also being developed for novel therapeutic interventions.3–5 While the heart’s left ventricle exhibits complex cellular alignment that underlies its global contractile function6,7, current methods for creating engineered cardiac tissues are largely restricted to thin sheets that possess uniaxial alignment. The ability to replicate more complex cellular alignment in engineered cardiac tissues may unlock new insights for drug discovery and disease modeling as well as more efficacious therapies. My Ph.D. thesis focuses on a novel biofabrication platform for creating stem-cell derived, cardiac tissues with programmable cellular alignment. Central to this platform is the scalable generation of cellularly aligned anisotropic organ building blocks (aOBBs), which are primarily composed of either iPSC-CMs or human dermal neonatal fibroblasts (hNDFs), the latter of which serves as a model system. Upon their release from micropillar arrays, these aOBBs are compacted into a bioink that contains gelatin and fibrinogen, which serves as the extracellular matrix (ECM). These densely cellular inks exhibit the requisite rheological properties for extrusion-based 3D bioprinting in both direct and embedded motifs. We locally encode cellular alignment within engineered cardiac tissues by aligning aOBBs along the print path during bioprinting. The shear and extensional flow fields that develop within printheads composed of tapered nozzles induce alignment of aOBBs, such that their long-axis aligns parallel to the print path during patterning of 1D filaments, 2D sheets, and 3D chambered structures. Upon printing, the desired cellular alignment is subsequently locked-in via polymerization of the surrounding ECM. Remodeling of the fibrin matrix facilitates the electromechnical fusion of individual aOBBs into a synchronously contractile tissue. However, cellular remodeling also generates stress lines that can reorient the prescribed cellular alignment. Through immunofluorescent staining, we demonstrated that cellular alignment persists in the core of printed cardiac filaments after 7 days of culture, while cardiomyocytes on the periphery of each printed filament reoriented in response to these passive stress lines. Reflecting this persistence of patterned cellular alignment, cardiac tissues printed from aOBB inks generated roughly three times higher force per cardiomyocyte compared to control tissues composed of cardiac spheroids. In addition, compared to cardiac tissue sheets printed perpendicular to the axis of passive stress, those printed parallel to the axis of passive stress generated higher force for all time points measured, up to 14 days, after bioprinting. Using aOBB inks, we fabricated 1D, 2D, and 3D aligned cardiac tissues via direct and embedded bioprinting. By embedded printing of aOBB inks within a sacrificial matrix, we have created three-dimensional tissues that exhibit programmed cellular alignment in arbitrary directions. As an exemplar, we produced a 3D tissue that mimics the helical cellular alignment present within the left ventricular myocardium. In summary, we can program arbitrarily complex alignment within 1D, 2D, and 3D cardiac tissues by first scalably generating aOBBs, then assembling these building blocks into densely cellular bioinks, and finally patterning these inks via direct and embedded bioprinting. Within days, aligned aOBBs within the printed constructs fuse into electromechanically synchronous cardiac tissues. Consequently, we can now directly program the magnitude and direction of force vectors within engineered cardiac tissues. Looking ahead, we envision combining this platform technology with vascularization methods to generate 3D chambered cardiac tissues with physiological alignment.