Recapitulating the spatial and temporal complexity of tissues or organs represents one of the biggest challenges in engineering tissue-like living constructs in vitro. Inkjet printing with its high positioning precision (±1μm) and reliable droplet generation (<5% variation) should fulfill the requirements to pattern biomaterials into complex three-dimensional (3D) geometries. However, there exists no biologically relevant ink that could afford 3D shape retention after drop deposition and fast stabilization of a printed structure. To address this issue, we have been developing an alginate hydrogel-based inkjet printing platform. Alginate solutions can be reliably dispensed on a hydrogel substrate storing calcium ions that quickly diffuse into the alginate solution inducing rapid cross-linking. We characterized the crosslinking kinetics and optimized printing parameters towards homogeneous stacking of droplets. Since living tissues are multi-component entities comprised of several types of cells and extracellular environments, we then focused on optimizing the dispensing system for synchronized multi-component deposition. We successfully matched the ejection characteristics of multiple nozzles by evaluating droplet diameter, jet speed and jet angle of each nozzle for variable dispensing voltage and pulse length.. Finally, proof-of-concept experiments were conducted to successfully print living 3D structures in the form of a simplified blood vessel
