Ref No: FBPR-5226
Date: 03-JAN-2023
Author: FS, VK, MSL. Version: 2

FRESH BIOPRINTING PROTOCOL

TeloCol-10
This is a suggested procedure, please adjust it according to your experimental needs.
To maintain the sterility of the product, work under sterile conditions.

Protocol aim

Materials needed

The aim of this protocol is to provide instructions forthe bioprinting of complex 3D structures with
TeloCol®-10 (Type I Collagen from bovine) Solution
from Advanced BioMatrix) using FRESH printing
method. It covers the steps of pre-print procedures,
printing, and post-print crosslinking. Changing theparameters in the protocol might change printing
conditions such as pressure and speed. Thisprotocol was optimized for the Temperaturecontrolled printhead using the BIO X.

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TeloCol®-10 (10 mg/mL)*
1X PBS and 10X PBS
Sodium hydroxide, 1M NaOH
LifeSupportTM*
Well plate or Petri dish*Temperature-controlled printhead*
BIO X* or BIO X6* bioprinterPositive displacement pipette
Eppendorf tube 1.5 mL
Ice bath
Cell suspension in cell culture medium
Cartridge, 3cc*
22G Conical bioprinting needle (1-inch length)**The product can be purchased in the CELLINKshop at www.cellink.com/shop.

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Protocol
This protocol is adjusted for printing scaffolds at the final TeloCol® concentration of 6 mg/mL. For otherconcentrations, recalculations need to be made but the same protocol can be followed. To avoid prematurecollagen self-assembly, we recommend keeping the collagen, all reagents (including cell culture medium) and all
consumables in the fridge prior to printing. Once compacted, LifeSupport™ should be kept in a fridge at 4°C andused within 12 hours. The ambient temperature should not exceed 23°C during handling or printing.

1.

Preparing LifeSupportTM bath

MATERIAL
LifeSupportTM
1X PBS
Well plate or Petri dish

DESCRIPTION• Add 40 mL of cold 1X PBS (4°C) to LifeSupportTM tube (sterile powder).
Note: 2 g of sterile LifeSupportTM powder corresponding to 15 mL of LifeSupportTM bath. For detailed directions,visit https://www.cellink.com/wp-content/uploads/2022/03/FluidformDirectionsforUseVersion6-rev-Sep-2021.pdf.
• Vortex for 1 minute.
• Put the tube into the fridge (4°C) for 15 minutes.
• Centrifuge for 5 minutes at 400 g.
• Gently pour off or aspirate the liquid supernatant.
• Grab the tube by the cap, hold it horizontally, and gently tap it against a palm 15 times.
• Shake the tube with dislodged LifeSupport™ vigorously for 10 seconds. Shake along the length of the tube.
• Centrifuge for further 5 minutes at 400 g.
• The LifeSupport™ should now be compacted at the bottom of the centrifuge tube. Gently pour off or aspirate
any remaining liquid supernatant to leave only the compacted LifeSupport™ in the bottom of the tube.
• Transfer the resulting LifeSupportTM bath with a sterile spatula into well plates or Petri dish and store it in afridge until use.

2.

Preparing for printing

MATERIAL
Temperature-controlled printheadBIO X or BIO X6 bioprinter
TeloCol®-10 (10 mg/mL)
Eppendorf tube 1.5 mL
Positive displacement pipette
Ice bath
10X PBS
1M NaOH sterile
Cell suspension in cell culture medium of choice
Cartridge, 3cc
22G Conical bioprinting needle (1-inch length)www.cellink.com

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DESCRIPTION• Place the Temperature-controlled printhead into the fridge at least 30 minutes before printing.
• Prepare the cell suspension, place in the fridge while preparing the TeloCol® solution.
• To prepare 1 mL of 6 mg/mL TeloCol® solution for printing, transfer 600 μL of TeloCol® 10 mg/mL solution
into a sterile Eppendorf tube using a positive displacement pipette. Keep the Eppendorf tube on ice.
• Add 100 μL of 10X PBS and use the same pipette to homogenize the resulting solution.
• Add 7.0 μL of 1M NaOH sterile. Pipette the solution up and down to neutralize collagen.
Note: The amount of NaOH needed to neutralize the TeloCol® 10 mg/mL can slightly vary from batch to batch.
We recommend the addition of it in small volume increments, adjusting the pH to 7.0-7.4.
• Add 293 μL of cell suspension and pipette the solution up and down until complete homogenization.
Note: The volume of cell suspension can change depending on the volume of NaOH needed for theneutralization. Adjust it to have a final volume of 1 mL bioink.
• Take the Temperature-controlled printhead from the fridge and mount on the BIO X or BIO X6, set thetemperature to 5°C and for the print bed 10°C, this is to guarantee the LifeSupportTM bath stability.
Note: Make sure the ambient temperature in the lab is maintained at 21-23°C, otherwise you may use ice packs
inside the printing chamber to prevent the printing area from overheating.
• Load a cartridge with the bioink using a pipette. Cap the cartridge with a printing needle and place it in theprinthead.

3.

PrintingMATERIALBIO X or BIO X6 bioprinterWell plate or Petri dish previously filled with LifeSupportTM bath
Cartridge with the TeloCol® bioink

DESCRIPTION• Place the well plate or Petri dish previously filled with LifeSupport TM bath and place it on the print bed. Printconstructs according to application. Suggested starting parameters are 3.5 mm/s printing speed and 8-10 kPa
pressure.
Note: If printability is not as desired, adjust the pressure and/or speed to up/down to extrude more/less material
at different speeds.

4.

Incubation and crosslinking

MATERIAL
Cell culture medium
DESCRIPTION• Keep the constructs for 10 min at room temperature to ensure initial collagen self-assembly prior to themelting of supporting bath.
• Incubate the constructs for 30 minutes at 37°C (5% CO2 and 95% relative humidity) for further self-assemblyof TeloCol® and LifeSupportTM melting.
Note: Large volumes may require longer times for the supporting bath to fully melt.
• Remove melted LifeSupportTM by replacing it with warm cell media to avoid handling the printed construct.
For example, if you printed into a 6-well plate, this can be done by carefully aspirating 2 mL of melted
LifeSupport™ out and adding 2 mL of warm cell media. Repeat this process until most of the support bathhas been replaced by media.

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