Layered Tissue Bioprinting Protocol

skin bioprinting tissue layering dermal layering collagen bioprinter


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.


  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

Pressure (PSI) Crosslink (sec)Print Temp (°C)


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.

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