Three-dimensional (3D) bioprinting presents a transformative approach to replicating vivo-like environments for mammalian cell cultures, offering potential advances in bioproduction and tissue engineering. In this study, we investigated the growth, metabolic activity, and structural organization of four mammalian cell lines (HEK, MDCK, CHO, and Vero) in 3D bioprinted constructs. Our results demonstrate that even highly selected, immortalised cell lines can regain physiological traits closer to their native tissue when cultured in 3D environments. We observed significant shifts in proliferation kinetics, including reduced growth rates and reduced fermentative activity. A Design of Experiment (DOE) approach identified critical biofabrication parameters—such as hydrogel microporosity and consolidation conditions—that modulate cell behavior and proliferation in 3D matrices. These findings highlight the potential of 3D bioprinting not only for medical applications, such as regenerative medicine and drug testing, but also for enhancing bioproduction processes by supporting higher cell densities and metabolic efficiency. Our work underscores the importance of optimizing 3D culture conditions to mimic vivo-like behaviors and improve productivity, offering new insights into the scalability of bioprinted constructs for industrial applications.