3D digital light processing bioprinting and in situ differentiation of hiPSCs for engineering cardiac tissue

The lack of physiologically relevant, reliable, scalable human cardiac tissue models remains a major barrier in cardiovascular disease research and drug development. Conventional 2D culture systems and animal models fail to accurately recapitulate the complex structure and function of the human myocardium, while current 3D strategies utilizing pre-differentiated cells face challenges in cell survival, integration, and tissue maturation. To address this critical gap, we developed FiBGel, a photocrosslinkable, choline bio-ionic liquid (BIL)-functionalized cold water fish gelatin methacryloyl (GelMA), optimized for direct 3D bioprinting of undifferentiated human induced pluripotent stem cells (hiPSCs) and subsequent in situ cardiac differentiation. FiBGel offered tunable mechanical and electrochemical properties, high cytocompatibility, and printability on a 3D digital light processing (DLP) bioprinter. For the first time, we report 3D DLP bioprinting of hiPSCs within FiBGel bio-ink, resulting in functional 3D cardiac tissue constructs. In contrast to 2D pre-differentiated cardiac cell encapsulation, in situ 3D cardiac differentiation of encapsulated hiPSCs in FiBGel constructs exhibited superior cardiac biomarker expression, sustained synchronous contractility, and long-term viability up to 4 months. This platform represents a significant advancement in stem cell-based biofabrication strategies for generating functional human tissue models for pharmacological screening, personalized disease modeling, and regenerative therapies. STATEMENT OF SIGNIFICANCE: This study reports the first ever demonstration of 3D digital light processing (DLP) bioprinting of undifferentiated human induced pluripotent stem cells (hiPSCs) encapsulated within a choline bio-ionic liquid-functionalized gelatin methacryloyl hydrogel, termed FiBGel. The bioprinted hiPSCs underwent in situ cardiac differentiation into functional engineered cardiac constructs. Unlike traditional methods reliant on pre-differentiated cell encapsulation, this strategy enables superior structural organization, sustained contractility, and viability over four months, an unprecedented duration for hiPSC-derived millimeter-scale cardiac constructs without external stimulation or perfusion. FiBGel combines tunable stiffness, electrochemistry, and cytocompatibility, addressing key limitations of current bio-inks. This work integrates stem cell biology, advanced biomaterials, and bioprinting to establish an in vitro system with strong potential for drug screening, disease modeling, and regeneration.