Ref No: FBPR-5201-5270-5271
Date: 03-JAN-2023
Author: FS, VK, MSL. Version: 2FRESH BIOPRINTING PROTOCOL

PhotoCol
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
PhotoCol® (Methacrylated Type I Collagen from
Advanced BioMatrix) using FRESH printing method.
It covers the steps of pre-print procedures, printing,andpost-printcrosslinking.
Changing
theparameters in the protocol might change printing
conditions such as pressure and speed. Thisprotocol was optimized for the pneumatic
Temperature-controlled printhead using the BIO X.

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PhotoCol®* (Kit 2,3,4* also includes 20 mM
acetic acid, Neutralization Solution and aphotoinitiator)
LifeSupportTM*
1X PBS
Well plate or Petri dishTemperature-controlled printhead*
BIO X*, BIO X6* 3D bioprinterSterile filter, 0.22 µm
Eppendorf tube 1.5 mL
Positive displacement pipette
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 PhotoCol® 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 PhotoCol® 10 mg/mL stock solution

MATERIALPhotoCol® (lyophilized methacrylated Type I collagen)20 mM acetic acid

DESCRIPTION▪ In a separate container, weigh the desired mass of PhotoCol® for stock solution preparation. Store theremaining PhotoCol® material in a fridge.
▪ Add the corresponding volume of 20 mM acetic acid to the PhotoCol® container to achieve the target
concentration of 10 mg/mL.
Note. For example, to prepare 3 mL of 10 mg/mL stock solution, add 3 mL of 20 mM acetic acid to 30 mg oflyophilized Photocol®.
▪ Mix on a shaker table or rotator plate at 2-10°C overnight or until fully solubilized.

2.

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 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 containing dislodged LifeSupport™ vigorously for 10 seconds. Shake along the length of thetube.
▪ 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 a
fridge until use.

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3.

Preparing for printing

MATERIAL
Temperature-controlled printheadBIO X or BIO X6 bioprinterPhotoinitiator (PI)
Neutralization Solution (NS)
Sterile filter, 0.22 µm
PhotoCol® 10 mg/mL stock solution
Eppendorf tube 1.5 mL
Positive displacement pipette
Cell suspension in cell culture medium of choice
Cartridge, 3cc
22G Conical bioprinting needle (1-inch length)

DESCRIPTION▪ Place the Temperature-controlled printhead in the fridge at least 30 minutes before printing.
▪ Prepare the cell suspension, place in the fridge while preparing the PhotoCol ® solution.
▪ Weigh the necessary amount of PI to achieve a desired concentration of PI in the final bioink.
Note: LAP is commonly used PI, and the suggested concentration is 0.25% (w/v) (see Table 1).
Table 1. Suggested values for the preparation of 1 mL of 6 mg/mL PhotoCol® bioink.
VbioinkCfinal bioink

Cstock solution

Vstock solution

VNSmPIVcell suspension

1 mL

6 mg/mL

10 mg/mL

600 μL45 μL

2.5 mg355 μL▪ Dissolve the PI in the NS.
Note: Always prepare some extra solution to compensate losses during filtration and transfer from one container
to another.
▪ Sterile filter the NS/PI solution using a 0.22 µm filter.
▪ To prepare 1 mL of 6 mg/mL PhotoCol® solution for printing, transfer 600 µL of PhotoCol® 10 mg/mL stock
solution into a sterile Eppendorf using a positive displacement pipette. Keep the reagents cold by keeping thetubes on ice.
▪ Add 45 μL of cold NS/PI solution to the PhotoCol® and use the same pipette to homogenize the resulting
solution. Place the tube on ice while setting up the BIO X.
▪ 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.
▪ Add 355 μL of cool cell suspension with desired cell density and pipette the solution up and down until
complete homogenization. Load a cartridge with the bioink using a pipette. Cap the cartridge with a printing
needle and place it in the printhead.

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4.

Printing

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

DESCRIPTION▪ Place the well plate or Petri dish previously filled with LifeSupportTM bath and place it on the print bed. Printconstructs using suggested parameters:•pressure at 8-10 kPa.

•speed at 3.5 mm/s.

Note: If printability is not as desired, adjust the pressure and/or speed to up/down to extrude more/less material
at different speeds.

5.

Incubation and crosslinking

MATERIAL
Cell culture medium
BIO X or BIO X6 bioprinter with LED modules of choice for photocuring
DESCRIPTION▪ Keep the constructs for 10 min at room temperature to ensure initial collagen self-assembly prior to themelting of the supporting bath.
▪ Incubate the constructs for 30 minutes at 37°C (5% CO2 and 95% relative humidity) for further self-assemblyof PhotoCol® and LifeSupportTM melting.
Note: Large volumes may require longer times for the supporting bath to fully melt.
▪ Photocure each construct using the LED module. Time will depend on a chosen PI and its concentration.
Note: It is recommended to use the 405 nm photocuring module for 30 seconds instead of 365 nm if possible,
when photocuring PhotoCol® with LAP photoinitiator. Overexposure at the 365 nm wavelength might damage
the cells.
▪ 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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