Lifeink 240

All collagen type 1 (Col1) was purchased as LifeInk 240, a pre-acidified collagen at 35 mg/mL (LifeInk240, Advanced Biomatrix). The urinary bladder matrix (UBM) was provided by the Badylak lab at 20 mg/mL concentrations in an acidified state. A 1:1 mixture of the Col1 and UBM was created by thoroughly mixing equal volumes of each component in two 10 mL BD syringes using a luer-lock syringe coupler. Approximately 4 mL of UBM and 4 mL of Col1 were mixed at a time and repeated until required bioink volume was created. This whole process was performed in a biosafety cabinet to maintain sterility. After mixing, the bioink was placed overnight in a temperature-controlled room at 21°C to bring the ink to room temperature before removing the air bubbles. By allowing the bioink go from 4°C to 21°C, it exposed more air bubbles to the surface that were condensed within the cold bioink. The next day, the resulting Col1-ECM ink was centrifuged at 3000 g for 10 min to remove bubbles. If needed, the inks were spun down for an additional to 5 min at 3000 g to remove excess bubbles. For printing, the ink was transferred to an ethylene oxide (EtO) sterilized 25 mL Gastight Syringe (Gastight Syringe, Hamilton Company).