00600500480.GOT
Date: 03-JAN-2023
Author: JH,VK. Version: 4

PHOTOCROSSLINKING OPTIMIZATION PROTOCOL

GelMA based bioinks
This is a suggested procedure, please adjust according to your experimental needs.

Protocol aim

Materials needed

The aim of this protocol is to provide instructions for
how to optimize the photocrosslinking of GelMA andGelXA based bioinks containing photoinitiators (PI)such as LAP or Irgacure. This protocol can be used
when recommended photocrosslinking procedure isnot sufficient or does not apply, for example at otherPI concentrations or dilutions.

•••••••

GelMA or GelXA based bioinks with PI*Water/PBS
BIO X*, BIO X6* or INKREDIBLE+* 3DbioprinterUV shielding cartridges, 3cc*
Conical bioprinting nozzles, 22-27G*
Well plate or Petri dish*Spatula*The product can be purchased in the CELLINKshop at www.cellink.com/shop.

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KEEP THE INK PROTECTED FROM LIGHT IF TRANSFERRED FROM THE ORANGE UV PROTECTEDCARTRIDGES TO AVOID CROSSLINKING BEFORE PRINTING. WORK WITH 3D PRINTERS IN DARK MODE.
THE PHOTOINITIATOR IS SENSITIVE TO REPEATED OR PROLONGED EXPOSURE TO HEAT.

ProtocolThis protocol works best using the BIO X or BIO X6 equipped with the Temperature-controlled printhead as wellas the cooled print bed. When using the INKREDIBLE+ system, pre-heat a printhead to 24-26°C to achieve thestable temperature maintenance. After deposition, printing substrates such as Petri dishes or well plates should beplaced on ice or another cooled surface to stabilize the construct prior to photocrosslinking.

1.

Preparing the bioinkMATERIAL
GelMA or GelXA based bioinks

DESCRIPTION•2.

Heat up the bioink bioink in the cartridge to 35°C until the bioink is liquid. The heating of the bioink canbe performed in a pneumatic or Temperature-controlled printhead or incubator.

Possible dilution

MATERIAL
Water/PBS

DESCRIPTION•3.

Simulate a cell suspension dilution or other dilution of the bioink with water or PBS. Mix in according toMixing cells and bioink Protocol.

Preparation for printing

MATERIAL
GelMA/GelXA in 3cc, UV shielding cartridges
Conical bioprinting nozzles, 22-27G

DESCRIPTION•

If the cartridge is taken directly from the heat, let sit for 10 min to cool down slightly. Observe any air
bubble movement, once it begins to slow down, the bioink is almost ready to print. The viscosity needs
to be like a thick syrup or honey.
•Place the semi-gelled GelMA/GelXA in either an INKREDIBLE+ printhead pre-heated to 24-26°C or theTemperature-controlled printhead on the BIO X/BIO X6 pre-heated to 24-26°C. Cap with the desired
printing nozzle. If using the BIO X/BIO X6, pre-cool the print bed to 15°C.
Note: The optimal printing temperature will depend on the bioink type and the bioink dilution.
•If proper viscosity and printability is not achieved by extending temperature equilibration time or tuningpressure:•
Too low viscosity, wide filaments despite using low pressure: decrease the printheadtemperature 0.5-1°C to increase the viscosity and equilibrate an additional couple of minutes.

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•4.

Too high viscosity, chunky filaments and high pressure required: increase the printheadtemperature 0.5-1°C to decrease the viscosity and equilibrate an additional couple of minutes.

PrintingMATERIALBIO X, BIO X6 or INKREDIBLE+ bioprinterWell plate

DESCRIPTION••5.

Bioprint several structures in a well plate according to your experimental needs or according to thatspecific bioinks Bioprinting Protocol.
Ensure that the bioprinted constructs are thermally gelled after printing by cooling the print bed if using
the BIO X/BIO X6 or placing the well plates containing printed construct on ice for 10 s if using theINKREDIBLE+.

Crosslinking optimization

MATERIAL
365/405 nm LED module for photocuring

DESCRIPTION•••If photocrosslinking during bioprinting, set the crosslinking parameters appropriately in the G-code for
the INKREDIBLE+ or the printhead setup page for the BIO X/BIOX6.
Choose relevant times and distances from light and constructs. Crosslink 1-3 constructs per chosenparameter.
Let the structure sit for 1-5 min to allow crosslinking after the light source is turned off.

Note: Bioink with LAP can be crosslinked using the 405 or 365 nm LED module. It is recommended to use
the 405 nm photocuring module instead of 365 if possible when using cells. Irgacure can only be crosslinked
with the 365 nm LED module.

6.

Incubation

MATERIAL
Water/PBS

DESCRIPTION••

7.

After photocrosslinking, add warm water or PBS in the wells to cover the constructs and agitate the platefor 2 min.
Incubate the constructs at 37°C for a few hours or overnight.

Crosslinking check

MATERIAL
Spatulawww.cellink.com

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DESCRIPTION•••

Check if the constructs are holding their shape by lifting the construct with a spatula.
Fill in the success rate according to Figure 1 of the constructs that hold their shape and those that has
dissolved.
Choose the successful crosslinking with the lowest time and distance for your experiment since
overexposure to the constructs might damage the cells.

CROSSLINKING TIME (S)DISTANCE FROM LED SOURCE (CM)

1530

45

60

7590

34567Figure 1. Example of a crosslinking success rate table that can be generated after testing different times anddistance from the construct.

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