Gelatine was functionalised as described before with small modifications to the original protocol (Shirahama et al., 2016 (link)). Ten grams medical grade 262 Bloom Type A gelatine (Gelita, Ter Apelkanaal, the Netherlands) was added to 1 L 0.25 M carbonate-bicarbonate (CB, Sigma-Aldrich, Zwijndrecht, the Netherlands) buffer pH 9.0 and allowed to dissolve at 50°C for 60 min. Heating was turned off and then 563 µL methacrylic anhydride (MAA; Sigma-Aldrich, CCID: 12974) was added dropwise in three steps. Ten minutes after each step the pH was measured and corrected using a 3 M NaOH (Sigma-Aldrich, CCID: 14798) solution to maintain pH 9.0. The gelatine-MAA solution was allowed to react overnight at room temperature (RT) while stirring. The next day the GelMA solution was dialysed using dialysis with molecular weight cut-off of 12–14 kDa (Sigma-Aldrich) for up to 5 days against deionised water pH 9.0. After dialysis, GelMA solution was aliquoted in 50 ml tubes, frozen with liquid nitrogen and lyophilised on a Labconco Freezone 2.5 L benchtop freeze-dryer (Labconco, Kansas City, MO, United States). Samples were considered dry when weights of the tubes were stable. Three batches were produced, lyophilised, re-dissolved into one large batch and then lyophilised again to minimise batch-to-batch variation.
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