Free-standing and support-bath-based direct 3D-bioprinting of hiPSC-cardiomyocytes

Cardiovascular diseases are the most common cause of death in the world. 3D-bioprinting is an emerging promising technology to generate human cardiac tissues in a controlled hierarchical structured manner. These biofabricated structures can be utilized either for direct clinical inventions, such as transplantation, or as heart models to study drug response or the effect of genetic mutations. Usually, cardiac tissues are casted. Recently the first approaches to 3D-print heart-like tissues have been reported. While casting does not allow the generation of complex hierachical structured tissues, 3D-bioprinting is in its infancy. Here, we present the first methods to either free-standing or support-bath-based direct 3D-bioprinting of human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CMs) to generate functional human heart ventricles that respond to pharmacological stimuli. For free-standing 3D-bioprinting, we have generated hydrogel microparticles from gelatin methacrylate (GelMA) utilizing the complex coacervation method. For support-bath-based 3D-bioprinting, we have utilized gelatin/gum arabic microparticles for the support bath and a collagen/hyaluronic acid-based bioink. Mechanical properties of bioinks and support-bath as well as electroconductivity were determined and the biocompatibility of the microparticles/bioinks was tested with NIH3T3 and/ or hiPSC-CMs before cardiac rings and ventricles were 3D-bioprinted by directly resuspending hiPSC-CMs within the bioinks. Accuracy and reproducibility of printing was analyzed via overlap-maps. Finally, printed cardiac tissues were analyzed by immunofluorescence analysis, calcium flux analysis using Fluo-4, pillar deflection, and/or drug responsiveness. These methods allow to directly 3D-bioprint hiPSC-CMs into functional human cardiac tissues in the shape of rings (2 mm diameter) and ventricles (8 mm long). Furthermore, stable multi-layered constructs and constructs with defined defects could be fabricated in an accurate and reproducible manner. The printed tissues had a wall thickness of 0.5 mm and live and dead staining images showed that fibroblasts as well as hiPSC-CMs survived the printing process with the same overall viability as when casted. Notably, hiPSC-CMs were evenly distributed throughout the biofabricated cardiac tissues and contained well-organized sarcomeres. These tissues exhibited spontaneous and regular contractions, which persisted up to several months and were able to contract against passive resistance. Importantly, beating frequencies of the printed cardiac tissues could be modulated by adrenergic pharmaceutical treatments, showing the potential of the biofabricated cardiac tissues as human heart models for drug screening/evaluation. Collectively, we demonstrate that it is possible to directly 3D-bioprint hiPSC-CMs. While there are still many challenges to overcome, the here developed methods open up new possibilities for generating complex functional heart tissues. For example, additional cell types such as fibroblasts or vascular cells can be integrated, cardiomyocytes from patients with a genetic disease can be used, and the complexity can be increased by 3D-bioprinting multi-chambered hearts and integrating valves. Taken together, our method provides a step towards the fabrication of advanced drug screening models, tissue grafts, and in the far future maybe even an engineered heart for organ transplantation.