Introduction: 3D-bioprinting is a promising technology to generate geometrically controlled hierarchical structures of human tissues. However, direct printing of living cells, especially in the case of soft tissues, has proven difficult. The evolution of in-support bath printing has provided significant advances in this field to take first steps towards generating anatomically accurate simplified heart models.[1-3] However, scaling up the support bath production and required steps in removing the support path pose challenges to this approach. Here, we present a method to directly and free-standing 3D-bioprint functional cardiac rings and ventricle-shaped cardiac tissues in an accurate and reproducible manner that can be cultured for at least one month and respond to pharmacological stimuli.
Methodology: We have generated hydrogel microparticles from gelatin methacrylate (GelMA), GelMA-- Poly (3,4-ethylenedioxythiphene) poly styrene sulfonate (PEDOT:PSS), and GelMA-polyethyleneimine-coated gold nanoparticles (bPEI-AuNPs)[2] utilizing complex coacervation method (Figure 1). We have analyzed particle size and rheological properties and electrical conductivity of each bioink. The biocompatibility of microparticles was tested with NIH3T3 and human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes before we 3D-bioprinted cardiac rings and ventricles by directly resuspending hiPSC-derived cardiomyocytes within hydrogel bioinks.
Figure 1. Schematic illustration of different microparticle bioinks.
Results: We have shown that based on the reaction criteria it is possible to control particle size in each bioink. More importantly, we have optimized printing approaches for the nozzle dimensions and type, achieving the best printing reproducibility with 27G tapered tips and a particle size of 40 – 70 µm. Live and dead staining images showed that fibroblasts (day 1) and hiPSC-derived cardiomyocytes (day 4) survived the printing process with a significant overall viability. Moreover, immunostaining images of hiPSC-derived cardiomyocytes showed well striated sarcomeric apparatus in these cells which were wrapping around particles. The 3D-bioprinted constructs started to beat after 3 days and maintained their contractile function during the 28 days of analysis. Importantly these tissues responded to adrenergic pharmaceutical treatments, showing their potential to be used as a model of cardiac tissue.
Conclusion: We have developed a novel approach to generate different types of microparticle hydrogels and controlled their morphological and dimensions. These bioinks provide a significant step forward in 3D-bioprinting of cardiac and potentially other electrically sensitive tissues and make it possible to directly free-standing 3D-bioprint hiPSC-derived cardiomyocytes to generate models of human cardiac tissues.
References:
[1] Esser et al. Adv Mater 2023
[2] Lee et al. Science 2019
[3] Noor et al. Adv Sci 2019
[2] Roshanbinfar et al. Adv Healthcare Mater 2023
