Ref No: BPR-IK-D12000
Date: 03-JAN-2023
Author: EZ,VK,JH. Version: 2

NEUTRALIZATION AND CASTING PROTOCOL

Collagen PREMIUM
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 forneutralization and casting of the Collagen PREMIUM
using the INKREDIBLE, INKREDIBLE+, BIO X orBIO X6, with and without cells. It covers bothdispensing of the biomaterial with encapsulated cells
and post seeding of cells on casted gels. Collagen
PREMIUM is crosslinked through thermal inducedgelation. This protocol was optimized for undiluted
Collagen PREMIUM as well as for a 10+1 cellsuspension diluted version. Changing theconcentration of solution to cell suspension ratio willchange the gelation time.

•••
••
•••••

Collagen PREMIUM*
Collagen PREMIUM Neutralization Solution*
Ice bath
3 mL syringes with Luer lock connection
Female/female Luer lock adaptor*
Cells* + culture medium*
Cartridges, 3cc*
Conical bioprinting nozzles*
BIO X*, BIO X6* or INKREDIBLE series* 3DbioprinterWell plate or mold*The product can be purchased in the CELLINKshop at www.cellink.com/shop.

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Protocol
This protocol works best with the BIO X/BIO X6 and the Temperature-controlled printhead. If using theINKREDIBLE+ system, the dispensing procedure should be performed fast to prevent the solution from warming
and gelling in the cartridge prior to dispensing.

1.

Cooling of material

MATERIAL
Collagen PREMIUM
Neutralization Solution
Ice bath

DESCRIPTION•

2.

Place the bottle of Collagen PREMIUM and the bottle of Neutralization Solution on ice to keep cool.

Neutralization

MATERIAL
3 mL syringes, two units
Female/female Luer lock adaptor

DESCRIPTION•Transfer equal volumes of Collagen PREMIUM and Neutralization Solution into two different 3 mLsyringes.

Note: Remove the syringe plunger, cap the syringe with a tip cap and pour the solution in the syringe. Insert
the plunger, flip the syringe and release the tip cap to evacuate the air.
•••3.

Connect the syringes via a female/female Luer lock adaptor and mix thoroughly back and forth. Be
careful not to introduce air bubbles.
Work fast to prevent materials from warming.
If not casting with cells or if seeding cells on-top, move directly to Step 4.

Mixing Collagen PREMIUM with cells

MATERIAL
Cell suspension in syringe
Cooled Collagen PREMIUM solution in syringe
Female/female Luer lock adaptor

DESCRIPTION•

Mix ten parts of cooled Collagen PREMIUM solution with one part of cell suspension without introducing
air bubbles to the mixture. For detailed instructions see the Mixing cells with bioink Protocol.
••

Attach the Collagen PREMIUM solution syringe to the syringe with cell suspension, using afemale/female Luer lock adaptor.
Carefully mix the solutions with the cell suspension by gently pushing the solutions back andforth between the syringes.

Note: Suggested cell suspension density is 1x106 cells/mL to 5x106 cells/mL of Collagen PREMIUM.

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Note: To avoid an air gap when mixing the solution and the cell suspension, carefully pre-fill the Luer lockadaptor with Collagen PREMIUM solution before attaching the syringe with the cell suspension.

4.

Preparing the bioink

MATERIAL
Cartridge, 3cc
Conical bioprinting nozzles

DESCRIPTION••

5.

If using the BIO X, pre-cool the Temperature-controlled printhead to 15°C and pre-heat the print bed to37°C. If using the INKREDIBLE-series, cool down the cartridge for 5 min if needed.
Transfer the solution (with or without cells) to the cartridge and cap the cartridge with a bioprinting
nozzle of choice. Place it in the printhead and connect the cartridge to the air adaptor.

CastingMATERIALBIO X, BIO X6 or INKREDIBLE series bioprinterWell plate or moldDESCRIPTION•

Dispense the required volume of solution in a mould or well plate.

Note: If waiting too long between extrusions, the solution can warm in the nozzle causing it to clog. If thisoccurs, replace with new nozzle.

6.

Thermal crosslinking

DESCRIPTION•Collagen PREMIUM thermally crosslinks.
•Incubate the constructs at 37°C until gelation occurs, approximately 10-15 min for volumes
below 100 µL. For larger constructs, prolong the incubation to 30 min. The BIO X/BIO X6 print
bed heated to 37°C can also be used for the thermal gelation.

Note: To verify that the crosslinking is sufficient, add 37°C media to one construct and observe that it doesn’t
dissolve.
•7.

If not seeding cells on-top of the casted collagen, move directly to Step 8.

Cell seeding

MATERIAL
Cell suspension

DESCRIPTION•

Dispense the cell suspension in the middle of the casted hydrogel. Suggested cell suspension density:
2x104 cells/cm2 to 5x104 cells/cm2. A highly concentrated cell suspension is suggested.

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8.

Incubation

MATERIAL
Cell culture medium

DESCRIPTION••

Add the desired medium to submerge the constructs. If cells were seeded on-top, wait for 1-2 h or until
the cells have started to attach to the collagen surface before submerging the constructs in medium.
Incubate the constructs in cell culture medium in standard culture conditions (37°C, 5% CO 2 and 95%
relative humidity) or according to your application.

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