Collagen and FRESH Bioprinting

Figure 1: Live/dead image of human neonatal dermal fibroblasts encapsulated in LifeInk200 (collagen) after 1 day of culture.

Figure 1: Live/dead image of human neonatal dermal fibroblasts encapsulated in LifeInk200 (collagen) after 1 day of culture.


LifeInk200 is a high concentration collagen bioink that can be thermally crosslinked at temperatures above 10ºC. Due to its concentration, it has impressive shape fidelity during bioprinting. However, since LifeInk200 cannot support itself before crosslinking, we recommend using the FRESH method as sacrificial support.

Storage and Handling

  • LifeInk200 should be stored at 4ºC in an airtight container.

  • IMPORTANT: All collagen handling should be done at 2-10ºC

You Will Need

In addition to LifeInk200 and FRESH slurry kit, you will also need:

  • Optional: well plate or printing dish of choice (FRESH kit comes with a 6 well plate)

  • Optional: transwell plates can help avoid sample loss or damage throughout the experiment. Keep in mind that transwell membranes may inhibit imaging processes.

  • Sterile 1mL BD syringe

  • Syringe coupler

  • Spatula

  • Ice


Instructions for Use

Part 1: Prepare Reagents


1a. Prepare LifeInk200.

In a sterile environment, attach the stock syringe of LifeInk200 to the coupler. Push the collagen up to the end of the coupler. Attach an empty 10mL syringe to the other end of the coupler. Make sure the plunger and cap are already all the way down. Inject the desired amount of collagen from the stock into the empty syringe. Decouple the stock syringe and return it to 2-10ºC. Set the new syringe aside with the coupler still attached.

1b. Mixing in cells.

Resuspend the cells in 200uL of media. Draw up the cell solution into a 1mL syringe. Attach the cell syringe to the other side of the coupler. Push the collagen into the cell syringe and continue to push back and forth until cells are thoroughly mixed in (~40 times). End with the cell-laden collagen in the 10mL syringe. Decouple the 1mL syringe, leaving the coupler attached to the 10mL syringe.

1c. Fill print syringe.

Using the second 10mL syringe, remove the plunger with the attached cap. Remove the cap from the plunger, place the cap back in the syringe opening, and push it down to the bottom with the plunger. This will ensure no air gets into the syringe. Make sure that the plunger doesn’t reattach to the cap.

Attach the print syringe to the open end of the coupler. Push all of the cell-laden collagen into the empty syringe. Decouple the newly filled syringe and attach needle.

Part 2: Print


2a. Design Files.

For the older software version (Bioprint Basic), prepare files through repetier host software, then upload the sliced gcode file to Allevi software. For the new software (Bioprint Pro), prepare your CAD file and upload an .stl to the platform for preparation.

2b. Load the LifeInk200 into the extruder.

Place your print container in the bot and calibrate the collagen to the plate. Before calibrating the empty extruder, remove the container and fill it with FRESH. Replace the container and calibrate the last extruder. Start your print!

2c. *After printing your designs, crosslink the finished structures at 37ºC for 30min – 1 hour.

After crosslinking, the melted FRESH must be removed. This can be a painstaking process as one needs to keep the structures submerged to provide support. Pipette some sterile media into the container. Then remove some of the FRESH-media mix. Continue to do this until the container is entirely filled with media.

*Crosslink times of 2 hours or higher can significantly decrease viability.


Print Settings

Extruder Speed (mm/s) Layer Height (mm) Nozzle Diam (mm) Gauge Pressure Print Temp(C) Volume Fill (ml)
Lifeink 200 (20 mg/ml) 6.0 0.2 0.2 30 untapered 15.0 4-10 5

These materials do not need to be photocrosslinked, so all of the crosslink settings may be set to 0.


Suggested Groups for Bioprint Study

When planning a bioprinting study, it is always important to carefully choose controls. Here are the control and experimental groups that we use when planning a study.

Control groups suggested for 3D Bioprint Study with Lifeink 200 and FRESH.
Lifeink Concentration Cells (millions/ml) FRESH? Design Print/Pipet
2D (0 mg/ml) 5 No N/A Pipet
20 mg/ml 5 Yes Thin film Pipet
20 mg/ml 5 No Thin film Pipet
20 mg/ml 5 Yes Thin film Print

2D controls are useful when screening for potential issues with culturing or contamination. If this control demonstrates low viability, it implies that there is an issue with your cell culture that needs troubleshooting.

3D pipetted controls are a control for Lifeink200 cell encapsulation. Because this control is pipetted, it removes any variables involved in bioprinting that might affect viability. Pipetting into FRESH tests for potential contamination with the support material. And finally, the bioprinted thin films would show if there was a decrease in viability due to the printing itself.


  1. Gelation time and gel stiffness can be affected by varying the concentration of LifeInk200 and duration of the crosslinking time. For help adjusting print parameters please contact [email protected]

  2. A fill volume change of more than 2 ml may affect pressure settings.

  3. A lower gauge size or tapered gauge will require lower pressure, while a higher gauge will require higher pressure for extrusion. Lowering the gauge size will also generally lower resolution.