Biofabrication has been receiving a great deal of attention in tissue engineering and regenerative medicine either by manual or automated processes. Different automated biofabrication techniques have been used to produce cell-laden alginate hydrogel structures, especially bioprinting approaches. These approaches have been limited to 2D or simple 3D structures, however. In this chapter, a novel bioprinting technique is disclosed for the production of more complex alginate hydrogel structures. This was achieved by dividing the alginate hydrogel cross-linking process into three stages: primary calcium ion cross-linking for printability of the gel, secondary calcium ion cross-linking for rigidity of the alginate hydrogel immediately after printing, and tertiary barium ion cross-linking for the long-term stability of the alginate hydrogel in the culture medium.