Printability, Durability, Contractility and Vascular Network Formation in 3D Bioprinted Cardiac Endothelial Cells Using Alginate–Gelatin Hydrogels

Abstract

Background: 3D bioprinting cardiac patches for epicardial transplantation are a promising approach for myocardial regeneration. Challenges remain such as quantifying printability, determining the ideal moment to transplant, and promoting vascularisation within bioprinted patches. We aimed to evaluate 3D bioprinted cardiac patches for printability, durability in culture, cell viability, and endothelial cell structural selforganisation into networks. Methods: We evaluated 3D-bioprinted double-layer patches using alginate/gelatine (AlgGel) hydrogels and three extrusion bioprinters (REGEMAT3D, INVIVO, BIO X). Bioink contained either neonatal mouse cardiac cell spheroids or free (not-in-spheroid) human coronary artery endothelial cells with fibroblasts, mixed with AlgGel. To test the effects on durability, some patches were bioprinted as a single layer only, cultured under minimal movement conditions or had added fibroblast-derived extracellular matrix hydrogel (AlloECM). Controls included acellular AlgGel and gelatin methacryloyl (GELMA) patches. Results: Printability was similar across bioprinters. For AlgGel compared to GELMA: resolutions were similar (200-700 µm line diameters), printing accuracy was 45 and 25%, respectively (AlgGel was 1.7x more accurate; p < 0.05), and shape fidelity was 92% (AlgGel) and 96% (GELMA); p = 0.36. For durability, AlgGel patch median survival in culture was 14 days (IQR:10-27) overall which was not significantly affected by bioprinting system or cellular content in patches. We identified three factors which reduced durability in culture: (1) bioprinting one layer depth patches (instead of two layers); (2) movement disturbance to patches in media; and (3) the addition of AlloECM to AlgGel. Cells were viable after bioprinting followed by 28 days in culture, and all BIO X-bioprinted mouse cardiac cell spheroid patches presented contractile activity starting between day 7 and 13 after bioprinting. At day 28, endothelial cells in hydrogel displayed organisation into endothelial network-like structures. Conclusion: AlgGel-based 3D bioprinted heart patches permit cardiomyocyte contractility and endothelial cell structural self-organisation. After bioprinting, a period of 2 weeks maturation in culture prior to transplantation may be optimal, allowing for a

Current state and future of 3D bioprinted models for cardio-vascular research and drug development

“…Three-dimensional cell cultures and technologies are rapidly gaining recognition for their potential to model heart tissue pathophysiology [4,[8][9][10][11]. Cells can be grown in scaffolds, scaffold-free or matrices environment aiming to mimic the ECM features of the heart; for example, biomaterial scaffolds, such as collagen and fibrin, provide a 3D environment for cells to attach, interact with each other and conduct electrical signals [4,6,7,12]. Cardiac myocytes cultured in 3D often employ a biomaterial such as a hydrogel or biocompatible polymer to mimic the ECM, providing a 3D architecture for cells to interact in all spatial dimensions, both with other cells and their environment.…”

“…Cardiac myocytes cultured in 3D often employ a biomaterial such as a hydrogel or biocompatible polymer to mimic the ECM, providing a 3D architecture for cells to interact in all spatial dimensions, both with other cells and their environment. The ability to modify properties such as elasticity, stiffness, conductivity and porosity allows for fine-tuning of the microenvironment [6,12]. These are core aspects of cardiac tissue engineering as the utility of 3D culturing and bioengineering to simulate blood flow, observe contractile forces and relaxation velocity in cardiac myocytes with variable mechanical and electrical cues are the tools necessary to create a complex and accurate microenvironment [6,12,13].…”

“…The ability to modify properties such as elasticity, stiffness, conductivity and porosity allows for fine-tuning of the microenvironment [6,12]. These are core aspects of cardiac tissue engineering as the utility of 3D culturing and bioengineering to simulate blood flow, observe contractile forces and relaxation velocity in cardiac myocytes with variable mechanical and electrical cues are the tools necessary to create a complex and accurate microenvironment [6,12,13]. The increase in parameters brings increased complexity and lack of standardized 3D culture protocols results in customized experimental design, which is not compatible with a high-throughput testing.…”

“…3D bioprinting of heart tissues combines additive manufacturing, biomaterials and viable objects to produce structured biological constructs which can be assembled and oriented. This new approach enables designed anisotropy for directed response, controls 3D cell composition and alignment mimicking native myocardium [4,6]. Extrusion-based bioprinters dispense cells embedded in hydrogels continuously in a pre-defined shape using either pneumatic or mechanical forces.…”

“…However, the lack of translation from the bench to the bedside, caused by limitations of currently available in vitro and in vivo models, has prompted a need to engineer novel tools for cardiovascular research. In the past two decades, 3D bioprinting technology has emerged as a potential tool to address limitations of conventional models by generating complex multicellular systems using cells and biomaterials A d v a n c e d o n l i n e a r t i c l e that better recapitulate the human heart microenvironment [4,6,7]. More recently, together with the use of stem cells, advances in the field of biofabrication enabled a move toward advanced in vitro models for drug testing.…”