Ref No: REP-IKG125000005
Date: 09-FEB-2023
Author: AB, VK, MG. Version: 2

DILUTION PROTOCOL

GelMA 20%
This is a suggested procedure, please adjust 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 on
how to dilute GelMA 20% (w/w) to your desiredconcentration using Reconstitution Agent P. The
obtained GelMA hydrogel can be used as a bioink on
its own or as a component in other bioink formulation.
Addition of a photoinitiator (PI) and use of 365 or 405
nm LED modules ensure stable and controlled
photocrosslinking of GelMA constructs for 3D cellculturing.

•••••••••

GelMA 20%*
Reconstitution Agent P* or an alternative bufferof choice
Photoinitiator* (PI)0.22 µm sterile syringe filter
Syringes with Luer lock connections
Female/female Luer lock adaptor*
Cartridges, 3cc*
Conical bioprinting nozzles*
BIO X*, BIO X6* or INKREDIBLE series* 3D
bioprinter*The product can be purchased in the CELLINKshop at www.cellink.com/shop.

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KEEP THE BIOINK 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.

Protocol1.

Calculations

DESCRIPTION•••••

Record the desired final concentration of GelMA (c F).
Record the desired final volume of GelMA bioink to prepare (VF).
Record the desired final concentration of PI (c PI_F). Common concentrations are between 0.01% and0.5%.
See Figure 1 for difference in temperature behavior of GelMA solutions at different concentrations.
Calculate the volume of GelMA 20% to be used.
𝑉𝐺𝑒𝑙𝑀𝐴20% =••𝑉𝐹 · 𝑐𝐹20%

See Table 1 for suggested cF.
Calculate the volume of Reconstitution Agent P, VR, to be used.
𝑉𝑅 = 𝑉𝐹 − 𝑉𝐺𝑒𝑙𝑀𝐴20%•Calculate the needed concentration of PI in the Reconstitution Agent P, cPI_R, to achieve your desired
final concentration of PI.
𝑐𝑃𝐼_𝑅 =•𝑉𝐹 · 𝑐𝑃𝐼_𝐹
𝑉𝑅

Calculate the amount of PI, mPI, needed to prepare VR and 1 mL extra to account for absorption in thesyringe filter.
𝑚𝑃𝐼 = 𝑐𝑃𝐼_𝑅 · (𝑉𝑅 + 1 𝑚𝐿)

Table 1. Suggested concentrations and the corresponding volume of GelMA 20% and Reconstitution Agent P usedfor the preparation of 5 mL of GelMA bioink.
Final concentration of GelMA,cF (%)

Volume of GelMA 20%, VGelMA20%(mL)

Volume of reconstitution agent
with PI, VR (mL)

5

1.25

3.75

102.5

2.5

2.

Preparing PI and Reconstitution Agent PMATERIALReconstitution Agent PPI
0.22 µm sterile syringe filter
Syringe with Luer lock connections

DESCRIPTION•

Dissolve mPI in VR + 1 mL of Reconstitution Agent P.

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•Sterile filter using a syringe and 0.22 µm sterile syringe filter into a container.

Note: Always remember to protect all PI containing solutions from light.

3.

Preparing GelMA bioink

MATERIAL
PI and Reconstitution Agent P
GelMA 20%
Syringes with Luer lock connections
Female/female Luer lock adaptor

DESCRIPTION••••

Transfer VR of the prepared Reconstitution Agent P with PI into a sterile syringe that can accommodate
minimum VF. Heat it to ~35°C.
Heat the GelMA 20% at ~35°C until it is liquid.
Transfer VGelMA20% of GelMA 20% into another syringe.
Connect the two syringes using a Luer lock adaptor, make sure there are no air bubbles present. Mix
the two solutions by passing them back and forth between the syringes until homogenized.

Note: If air bubbles are introduced into the mixture, centrifuge the heated solution at 1000 x g for 1 min toremove them.
Note: If not using the GelMA bioink right away, store at 4-8°C protected from light.

4.

BioprintingMATERIALGelMA bioink
Cartridges, 3cc
Conical bioprinting nozzles
BIO X, BIO X6 or INKREDIBLE+DESCRIPTION•Transfer the GelMA bioink to a cartridge and cap with a bioprinting nozzle. Place in the printhead andprint according to application.
••

For an example on bioprinting GelMA 10% with cells, see the Bioprinting Protocol GelMABioink.
For bioprinting GelMA 5% with cells, the Bioprinting Protocol GelMA FIBRIN can be used as areference with slight modifications in pressure and no thrombin crosslinking.

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Figure 1. Decrease of storage modulus for GelMA hydrogels at various concentrations over an increasing
temperature.

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