Synthesis and Characterization of Methacrylated Gelatin

The GelMA was synthesized as follows [22 ,23 (link),24 (link),25 (link)]. First, type-A porcine skin gelatin was added at 10% w/v to phosphate-buffered saline (PBS) and heated at 50 °C for 1 h with stirring. Methacrylic anhydride (Sigma-Aldrich Japan, Tokyo, Japan) was then added (7.5% v/v) to the gelatin solution at around 60 °C while stirring, allowing it to react for 2 h. In synthesized methacrylated gelatin, gelatin macromers containing primary amino groups were reacted using Methacrylic anhydride to add methacrylate pendant groups, as shown in Figure 1a. The samples were then dialyzed using 12–14 kDa cutoff dialysis tubing (Spectra/Por(R), Repligen, Waltham, MA, USA) in pure water for 1 week. Finally, the solution was frozen overnight (−80 °C), lyophilized for 1 week in a freeze-dryer (FDU-1200, Eyela, Tokyo, Japan), and stored at −80 °C until further use. The lyophilized GelMA was a white porous foam, as shown in Figure 1c. The lyophilized GelMA was dissolved in PBS or culture medium to fabricate GelMA microstructures.