The organ transplantation field requires new approaches for replacing and regenerating tissues due to the lack of adequate transplant methods. Three-dimensional (3D) extrusion-based bioprinting is a rapid prototyping approach that can engineer 3D scaffolds for tissue regeneration applications. In this process, 3D printed cell-based constructs, consisting of biomaterials, growth factors, and cells, are formed by the extrusion of bioinks from nozzles. However, extrusion applies shear stresses to cells, often leading to cellular damage or membrane rupture. To address this limitation, herein, we developed and optimized a 3D bioprinting approach by evaluating the effect of key extrusion-based 3D bioprinting parameters-bioink viscosity, nozzle size, shape, and printing speed-on cell viability. Our results revealed that cells printed in higher-viscosity bioinks, with smaller, cylindrical nozzles, exhibited lower viability due to their exposure to high shear stresses. Translational flow speed had a cell-dependent impact, as different cell types have different sensitivities to the magnitude and duration of shear stress inside the nozzle. Overall, evaluating these parameters could facilitate the development of 3D high-resolution bioprinted constructs for tissue regeneration applications, offering a more efficient alternative to traditional fabrication methods, which are often labor intensive, expensive, and repetitive.