Begin the preparation of the hydrogel bioink aseptically inside a biosafety cabinet by first preparing a 0.25 milligrams per milliliter stock solution of PEG alpha methacrylate in sterile cell culture media without FBS. Also, using 20-millimolar sterile TCEP, prepare 250-millimolar stock solutions of DTT, MMP2 degradable crosslinker, and the RGD peptide. Finally, prepare a 15 weight percent stock solution of PEO using distilled water.
Combine the required amounts of the prepared reagent stock solutions and low-serum cell culture media with the fibroblasts in a 15-milliliter conical tube. Mix the resultant bioink thoroughly using a positive displacement pipette to ensure the cells are single. Verify that the final precursor solution has a pH of 6.2.
Remove the print head from the bioprinter to access the glass syringe. To load the bioink into the glass syringe, remove the syringe plunger. Then, collect the bioink from the centrifuge tube using a separate syringe fitted to a 1.5-inch-long, 15-gauge, blunt-tip needle.
Replace the 15-gauge needle with a 30-gauge, 0.5-inch-long, blunt-tip needle and transfer it to the glass syringe, avoiding air bubble formation. Finally, place the glass syringe within the print head and attach the print head components while ensuring a firm assembly for printing.
