Fibroblast Bioprinting Protocol

Overview

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

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

Materials

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. Human neonatal dermal fibroblasts or human adult dermal fibroblasts
   • Note: you may also add keratinocytes and melanocytes
9. Fibroblast growth medium

Methods

1. Follow this protocol to prepare PureCol® and your cells for printing;
2. Set your Allevi extruder to 4˚C;
3. Attach the Allevi layering tip™ to your syringe;
4. 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;
5. Incubate your structure at 37˚C for at least 30 minutes; 
6. 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

We hope that you found this step-by-step protocol for skin bioprinting useful! You are now ready to image and analyze your 3D bioprinted skin construct – click here for a full list of analysis protocols.

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