How to Mix Native Protein Additives with Bioinks

Overview

Follow this protocol to learn how to mix native protein additives with bioinks for enhanced cellular response in 3D bioprinting applications.

Materials

• Allevi plastic syringes (5 mL or 10 mL)
• Cells
• Hydrogel (collagen, gelMA, alginate, hyaluronic acid, etc)
• Sterile syringe coupler
• Native protein additive (collagen type III, type IV & type V, fibronectin, tropoelastin, vitronectin, etc)

Methods: Mixing Native Protein Additives with Bioinks

1. Add the concentrated cell suspension in cell culture media to a sterile syringe.
2. Add desired neutralized protein (ie. Collagen type III) to cell suspension/cell culture media from step 1.
3. Place sterile coupler on the end of the syringe with the cell suspension.
4. Slowly push plunger in until media forms a slight external meniscus above the end of the coupler on the syringe.
5. Remove cap from the syringe with collagen and slowly push plunger in until collagen forms a slight external meniscus above the end of the syringe.
6. Couple the syringe with cells to the syringe with collagen. (Ensure that there are no air bubbles in the system. The “external meniscus” on both syringes helps ensure that there are no air bubbles introduced).
7. Slowly push plungers back and forth ~40 times to ensure thorough mixing. End with all of the material in the syringe to be used for printing.
8. The cell-laden bioink is now ready for positive displacement printers.

Note

• We recommend a final bioink cell concentration of 5X10^6/mL or greater.
• For best results, add 2 mL of cell suspension/additional protein per 5 mL of collagen bioink. Use a similar ratio for smaller volumes.
• For pneumatic printers, transfer the collagen into an appropriate syringe using the coupler. The new syringe should have the seal inserted, but the plunger removed. Centrifuge the syringe at 2000 RPM for 1 minute after transferring the collagen to remove any air bubbles.

We hope that you found this protocol helpful for learning how to mix native protein additives with bioinks! You are now ready to start bioprinting with your protein and cell-laden bioink. Click here to read more bioprinting protocols, tips, and tricks.