Ref No: BPR-IK-200011
Date: 02-JAN-2023
Author: EP,JH,VK. Version: 3BIOPRINTING PROTOCOLCELLINK A-RGD
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 forbioprinting of CELLINK A-RGD using the BIO X, BIO
X6, INKREDIBLE or INKREDIBLE+ bioprinter, andcovers steps from pre-print mixing with cells, 3D
bioprinting and post-print processes such as ioniccrosslinking. This protocol was optimized for
CELLINK A-RGD, undiluted as well as using a 10+1
cell suspension dilution. Changing the parameters inthe protocol might change the crosslinking timerequired. This protocol was optimized using thepneumatic printhead on the BIO X.

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CELLINK A-RGD*
Cells* + cell culture medium*3 mL syringes with Luer lock connections
Female/female Luer lock adaptor*
CELLMIXER* (optional)
Green or clear cartridges, 3cc*
Conical bioprinting nozzles*
BIO X*, BIO X6* or INKREDIBLE-series* 3D
bioprinterWell plate or Petri dish*Crosslinking Agent (included with the bioinkpurchase)*The product can be purchased in the CELLINKshop at www.cellink.com/shop.

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Protocol
This protocol can be performed with printheads and print bed at room temperature, where room temperature is
between 20-25°C.

1.

Preparing the bioink

MATERIALCELLINK A-RGD

DESCRIPTION••

2.

Warm up CELLINK A-RGD in a cartridge to room temperature.
If not printing with cells, move directly to step 3.

Mixing the bioink with cellsMATERIAL
3 mL syringes with Luer lock connections
Female/female Luer lock adaptor
Pre-warmed CELLINK A-RGD
Cell suspension
Cartridge, 3cc
CELLMIXER (optional)

DESCRIPTION•••

At this point, mix ten parts bioink with one part cell suspension, taking care not to introduce air bubbles
to the mixture. For detailed instructions see the Mixing cells with bioink Protocol.
If preparing for quantities < 2 mL of CELLINK A-RGD, it is recommended to connect two 3 mL Luer locksyringes, one with the bioink and the other with the cell suspension and gently mix back and forth
between the syringes until homogeneous. Transfer the mixture to an empty 3cc cartridge by connecting
the syringe to the cartridge using the Luer lock adaptor. Cap the cartridge with a tip cap.
If using larger quantities, the CELLMIXER can be used:•Transfer the cell suspension to the 1 mL cell syringe (PART 1) using a female/female Luer lockadaptor.
•Transfer the bioink to the 12 mL syringe (PART 2) using a female/female Luer lock adaptor.
•Clip both syringes to the Dispensing unit (PART 3).
•Connect the two syringes to the Mixing unit (PART 4), then connect the Empty cartridge (PART5) to the Mixing unit’s other side.
•Apply gentle pressure onto the Dispensing unit to mix the content of both syringes into theempty cartridge. Cap the cartridge with a tip cap.

Note: To avoid introducing air when connecting the syringes, carefully pre-fill the Luer lock adaptor with CELLINK
A-RGD before attaching it to the syringe with the cell suspension.

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3.

Preparation for printing

MATERIALCELLINK A-RGD mixed with cells (if applicable) in cartridge
Conical bioprinting nozzles

DESCRIPTION•

Cap the room tempered CELLINK A-RGD with a bioprinting nozzle of choice and place in the printhead.
Connect the cartridge to the air adapter.

Note: Be careful not to touch the printhead with the nozzle tip and if using very liquid materials, make surethat the bioink does not drip through the nozzle especially when attaching the air adapter. Alternatively, thecartridge can be placed in the printhead with the tip cap on and when in place switched to a nozzle.
Note: Test the flow of the bioink after the calibration is performed and start with a low pressure and increase
stepwise.

4.

PrintingMATERIALBIO X, BIO X6 or INKREDIBLE series bioprinterWell plate or Petri dish

DESCRIPTION•

Bioprint structures using suggested parameters (Table 1) on to a well plate or Petri dish. If printability is
not as desired, adjust the pressure up/down by 1 kPa to extrude more/less material.

Note: For printing complicated structures, it is recommended that CELLINK A-RGD is used in conjugation
with CELLINK START to aid in its stability or with the use of the Syringe Pump printhead to better control
extrusion rates.

Table 1. Recommended minimal extrusion pressure** (±2 kPa) used for printing continuous filaments at 20-25°C
with cells/
without cells. Again, ‘with cells’ assumes a mixture of one part cell suspension to ten parts bioink. For highly
concentrated cell suspensions, the pressure needs to be increased towards the pressure used for undiluted bioink.
Printing speed (mm/s) →5101520Nozzle size (G) ↓22325427

544634645845746946105711**Note this is only a recommended reference of starting pressures. The actual pressure needed will vary depending on thepreparation procedures (amount of bioink and actual temperature of the bioink) as well as the fitting of the piston in the cartridge
and the leveling of the print surface. This table was generated with bioink temperature of 24°C and with a 10+1 bioink dilution
with cell suspension.

5.

Crosslinking

MATERIAL
Crosslinking Agent
Cell culture medium

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DESCRIPTIONCELLINK A-RGD can be crosslinked with ions using the CaCl2-containing Crosslinking Agent.
•Submerge the cell-laden constructs in the Crosslinking Agent for 30 seconds to 5 minutes depending on
construct size, infill density and desired construct stiffness. Remove Crosslinking Agent and rinse
constructs with basal culture media once.
Note: 30 seconds is recommended for 10 µL droplets while 10 minutes might be required for dense 1 cm 3blocks. In addition, optimize the crosslinking depending on the cell type.

6.

Incubation

MATERIAL
Cell culture mediumDESCRIPTION••

After crosslinking and washing, add the desired medium to the construct and place in incubator.
Incubate the constructs in cell culture medium in standard culture conditions (37°C, 5% CO2 and 95%
relative humidity) or according to your application. Replace medium regularly.

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