Role of pH and Crosslinking Ions on Cell Viability and Metabolic Activity in Alginate-Gelatin 3D Prints

Alginate-gelatin hydrogels are extensively used in bioengineering. However, despite different formulations being used to grow different cell types in vitro, their pH and its effect, together with the crosslinking ions of these formulations, are still infrequently assessed. In this work, we study how these elements can affect hydrogel stability and printability and influence cell viability and metabolism on the resulting 3D prints. Our results show that both the buffer pH and crosslinking ion (Ca²⁺ or Ba²⁺) influence the swelling and degradation rates of prints. Moreover, buffer pH influenced the printability of hydrogel in the air but did not when printed directly in a fluid-phase CaCl₂ or BaCl₂ crosslinking bath. In addition, both U2OS and NIH/3T3 cells showed greater cell metabolic activity on one-layer prints crosslinked with Ca²⁺. In addition, Ba²⁺ increased the cell death of NIH/3T3 cells while having no effect on U2OS cell viability. The pH of the buffer also had an important impact on the cell behavior. U2OS cells showed a 2.25-fold cell metabolism increase on one-layer prints prepared at pH 8.0 in comparison to those prepared at pH 5.5, whereas NIH/3T3 cells showed greater metabolism on one-layer prints with pH 7.0. Finally, we observed a difference in the cell arrangement of U2OS cells growing on prints prepared from hydrogels with an acidic buffer in comparison to cells growing on those prepared using a neutral or basic buffer. These results show that both pH and the crosslinking ion influence hydrogel strength and cell behavior.