Skin Bioprinting Protocol

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Overview

Skin engineering is a prominent alternative to traditional methods used for wound healing, drug testing and discovery, and other skin-related applications. However, challenges remain when it comes to generating complex skin substitutes. 

Collagen is extremely difficult to pattern in optimal concentrations (3 mg/mL).  With Allevi’s exclusive CORE™ printhead and the Skin Kit, 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. Allevi’s Skin Kit presents a novel solution to automating and standardizing the creation of skin models.

Storage and Handling

Collagen should be stored  4˚C.  

You Will Need

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

Instructions for Use

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
50.20.227

Pressure (PSI) Crosslink (sec)Print Temp (°C)
104

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

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