Layered Tissue Bioprinting Protocol

Overview

Collagen type I is the most abundant protein in the extracellular matrix and has been widely used in academia and industry for tissue engineering applications. With Allevi’s exclusive CORE™ printhead and the layered tissue bioprinting protocol, you are now able to print and pattern pure 3 mg/mL type I collagen or 8 mg/mL methacrylated collagen. This is the first time that such low concentration collagen can be printed and patterned through 3D bioprinting. 

Storage and Handling

Collagen should be stored  4˚C. 

You Will Need

1. 10 mL of Advanced Biomatrix PureCol
2. Allevi 5 mL syringe
3. 2 x Allevi layering tips™
4. Syringe coupler
5. NaOH
6. 10X PBS
7. Cell media
8. Cells

All items come sterile. 

Instructions for Layered Tissue Bioprinting:

Collagen preparation:

1. Add 1 part of chilled 10X PBS to 8 parts of Advanced Biomatrix PureCol;
   1. Collagen should be maintained at a temperature of 4˚C to prevent premature crosslinking;
2. Adjust the pH of the mixture to 7 – 7.5 by adding sterile 0.1 M NaOH;
3. Add water to complete full volume to 10 parts;
4. Mix cells with collagen by pipetting collagen onto cell pellet;
5. With the Allevi syringe, draw 1.5 mL of your cell solution.

Printing:

1. Set your Allevi extruder to 4˚C;
2. Attach the Allevi layering tip™ to your syringe;
3. Bioprint on the Allevi printing dish;
   1. If you have an A3, set your bed plate to 37˚C to crosslink your skin layers at the time of deposition;
4. Incubate your structure at 37˚C for at least 30 minutes; 
5. Culture your layered tissue as desired.

Print Settings

Speed (mm/s)	Layer height (mm)	Nozzle Diam (mm)	Gauge
5	0.2	0.2	27

Pressure (PSI)	Crosslink (sec)	Print Temp (°C)
10	–	4

References

Lee, V., Singh, G., Trasatti, J. P., Bjornsson, C., Xu, X., Tran, T. N., … Karande, P. (2014). Design and Fabrication of Human Skin by Three-Dimensional Bioprinting. Tissue Engineering Part C: Methods, 20(6), 473–484. https://doi.org/10.1089/ten.tec.2013.0335