2.1. Fabrication of the GelMA Hydrogel

To fabricate the gelatin methacryloyl (GelMA) hydrogel, gelatin from porcine skin (Millipore Sigma-Aldrich) was chemically modified using methacrylic anhydride (MAAH) (Millipore Sigma-Aldrich) as described by Loessner et al [15]. Briefly, 10% gelatin was fully dissolved in Dulbecco’s phosphate-buffered saline (DPBS) (HyClone) at 50°C and 0.8 mL MAAH per gram of gelatin was added dropwise. The reaction was maintained in a water bath at 50 °C for 2.5 h and was stopped by diluting the mixture 1:3 using DPBS at 40 °C. The mixture was centrifuged at 3500 xg and dialyzed against deionized (DI) water over 1 week at 40 °C to remove unreacted MAAH. The dialyzed solution was lyophilized for 1 week into dry foam and stored at −20°C until used.

To make the pre-polymer solution of the hydrogel, GelMA foam was dissolved in DPBS at a final concentration of 10%, followed by 2.5% Irgacure 2959 (2-hydroxy-4’-(2-hydroxyethoxy)-2-methylpropiophenone) (Millipore Sigma-Aldrich) as described by Loessner et al [15].

The prepolymer solution of 250 μL per well was added to the 48-well cell culture plate and photo-crosslinked using ultraviolet (UV) light at 365 nm for 10 min at room temperature. The UV light was exposed from the top of the plate using a UVP UVL-21 Compact 4-Watt UV lamp. The hydrogels were then immersed in DPBS at 37 °C overnight to remove any uncrosslinked material. In order to store the hydrogels in a dry condition, they were frozen at −80 °C overnight and dehydrated by lyophilizing at −50°C for 3 days using a lyophilizer (Labconco). The dehydrated hydrogels were stored at −20°C before the following sterilization procedures and measurements were performed.