The use of fluid-phase 3D printing to pattern alginate-gelatin hydrogel properties to guide cell growth and behaviour in vitro.

3D (bio)printing technology has boosted the advancement of the biomedical field. However, tissue engineering is in its infancy and (bio)printing biomimetic constructions for tissue formation in vitro is still a default. As a new methodology to improve in vitro studies, we suggest the use of a cross-linkable aqueous support bath to pattern the characteristics of the scaffolds during the 3D printing process. Using fluid-phase, different molecules can be added to specific locations of the substrate promoting cell behaviour guidance and compartmentalization. Moreover, mechanical aspects can be customized by changing the type or concentration of the solution in which the (bio)printing is acquired. In this study, we first assessed different formulations of alginate/gelatin to improve cell colonization in our printings. On formulations with lower gelatin content, the U2OS cells increased 2.83 times the cell growth. In addition, the alginate-gelatin hydrogel presented a good printability in both air and fluid-phase, however the fluid-phase printings showed better printing fidelity as it diminished the collapsing and the spreading of the hydrogel strand. Next, the fluid-phase methodology was used to guide cell colonization in our printings. First, different stiffness were created by crosslinking the hydrogel with different concentrations of CaCl2 during the printing process. As a result, the U2OS cells were compartmentalized on the stiffer parts of the printings. In addition, using fluid-phase to add RGD molecules to specific parts of the hydrogel has also promoted guidance on cell growth. Finally, our results showed that by combining stiffer alginate-gelatin hydrogel with RGD increasing concentrations we can create a synergetic effect and boost cell growth by up to 3.17-fold.This work presents a new printing process for tailoring multiple parameters in hydrogel substrates by using fluid-phase to generate a more faithful replication of the in vivo environment.&#xD.