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Abstract Three-dimensional (3D) bioprinting has emerged as a powerful engineering approach for various tissue engineering applications, particularly for the development of 3D cellular structures with unique mechanical and/or biological properties. For the jammed gelatin microgel-gelatin solution composite bioink, comprising a discrete phase of microgels (enzymatically gelled gelatin microgels) and a cross-linkable continuous gelatin precursor solution-based phase containing transglutaminase (TG), its rheological properties and printability change gradually due to the TG enzyme-induced cross-linking process. The objective of this study is to establish a direct mapping between the printability of the gelatin microgel-gelatin solution based cross-linkable composite bioink and the TG concentration and cross-linking time, respectively. Due to the inclusion of TG in the composite bioink, the bioink starts cross-linking once prepared and is usually prepared right before a printing process. Herein, the bioink printability is evaluated based on the three metrics: injectability, feature formability, and process-induced cell injury. In this study, the rheological properties such as the storage modulus and viscosity have been first systematically investigated and predicted at different TG concentrations and times during the cross-linking process using the first-order cross-linking kinetics model. The storage modulus and viscosity have been satisfactorily modeled as exponential functions of the TG concentration and time with an experimentally calibrated cross-linking kinetic rate constant. Furthermore, the injectability, feature formability, and process-induced cell injury have been successfully correlated to the TG concentration and cross-linking time via the storage modulus, viscosity, and/or process-induced shear stress. By combing the good injectability, good feature formability, and satisfactory cell viability zones, a good printability zone (1.65, 0.61, and 0.31 h for the composite bioinks with 1.00, 2.00, and 4.00% w/v TG, respectively) has been established during the printing of mouse fibroblast-based 2% gelatin B microgel-3% gelatin B solution composite bioink. This printability zone approach can be extended to the use of other cross-linkable bioinks for bioprinting applications.more » « less