SABER bioprinting: a temporal platform for multiday assembly of engineered tissues.

Current bioprinting approaches are constrained to single-session fabrication, limiting the scale, temporal complexity, and biological maturation achievable in engineered constructs. Here, we demonstrate the feasibility of Sequential Additive Biofabrication Extended over Real-time (SABER), a bioprinting strategy that introduces time as an explicit design variable by enabling multi-day additive fabrication of soft-hydrogel constructs supported by continuous perfusion culture. SABER integrates a thermoresponsive methylcellulose-agarose support material, which allows high-fidelity collagen deposition at low temperatures and mechanical stabilization at culture temperatures, with a custom bioreactor that permits direct through-tissue perfusion. We demonstrate feasibility of multi-day fabrication of layered constructs, nested geometries, suspended internal features, a miniaturized acellular human heart model with preserved architectural fidelity, and a 10-layer approximately 1 cm thick construct demonstrating scalability beyond single-session practical limits. SABER supports perfusion culture of thick cell-laden constructs, enabling printed cardiac tissues to remain viable and contractile after one week of perfusion culture and permittingin situdifferentiation of bioprinted iPS cells into cardiomyocytes. These findings establish SABER as a proof-of-concept platform for time-resolved, perfused bioprinting of soft hydrogel constructs, introducing time as an explicit design variable in biofabrication and providing a foundation for future work exploring multi-day fabrication workflows at scales and with temporal complexity not supported by existing single-session approaches.