Ref No: BPR-IK-102010
Date: 02-JAN-2023
Author: EP, JH, VK. Version: 5

BIOPRINTING PROTOCOLCELLINK RGDThis 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 RGD using theINKREDIBLE, INKREDIBLE+, BIO X or BIO X6, andcovers steps from pre-print mixing with cells, 3D
bioprinting and post-print processes such as ioniccrosslinking. This protocol was optimized for
CELLINK 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.



CELLINK RGD*
Cells* + cell culture medium*3 mL syringes with Luer lock connections
Female/female Luer lock adaptor*
CELLMIXER* (optional)
Clear cartridges, 3cc*
Conical bioprinting nozzles, 22-27G
recommended*BIO X*, BIO X6* or INKREDIBLE-series* 3DbioprinterWell plate or Petri dish*Crosslinking Agent* (included with the bioinkpurchase)*The product can be purchased in the CELLINKshop at www.cellink.com/shop.

www.cellink.com

info@cellink.com

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 RGD
3 mL syringe with Luer lock connections
Female/female Luer lock adaptor
Pipette tip or spatula
DESCRIPTION•••2.

Mix the full 3 mL of CELLINK RGD a few times to make sure it is homogenous. Connect the cartridge toa 3 mL syringe using a Luer lock connector and remove the end cap. Push the bioink from the cartridge
into the syringe by gently pushing the cartridge piston using a pipette tip or small spatula while
simultaneously pulling the syringe plunger. Gently mix the bioink back and forth between the cartridgeand syringe to homogenize the bioink, taking care not to introduce air. If not using the entire 3 mL of thebioink in the cartridge, spare the rest of the bioink in the optimal storage conditions.
Warm up CELLINK RGD to room temperature. Only warm the needed volume.
If not printing with cells, move directly to step 3.

Mixing the bioink with cells

MATERIAL
3 mL syringes with Luer lock connections
Female/female Luer lock adaptor
Pre-warmed CELLINK RGDCell 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 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.

www.cellink.com

info@cellink.com

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

3.

Preparation for printing

MATERIALCELLINK RGD mixed with cells (if applicable) in cartridge
Conical bioprinting nozzles, 22-27G
DESCRIPTION•

Cap the cartridge with a printing nozzle of choice and place the room tempered CELLINK RGD in theprinthead. Connect the cartridge to the air adapter.

Note: The recommended nozzle size is 22G. Decrease the nozzle diameter to achieve smaller filament
diameter, however this also increase the risk of the bioink clogging.
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.

Printing

MATERIALBIO X, BIO X6 or INKREDIBLE series bioprinterWell plate or Petri dishDESCRIPTION•

Bioprint structures with parameters according to 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: If waiting too long between extrusions the bioink can dry in the nozzle causing it to clog. If this occurs,
replace with new nozzle.
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 and 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) →Nozzle size (G) ↓522625

827

10

10
11
12
13

8
10
14

15
13
15
15

9
1115

20
14
17
17

1112
16

17
1720

** This is only a recommended reference of starting pressures. The actual pressure needed will vary depending on the preparation
procedures (amount of bioink and actual temperature of the bioink) as well as the fitting of the piston in the cartridge and theleveling of the print surface. This table was generated with bioink temperature of 22°C and with a 10+1 bioink dilution with cellsuspension.

www.cellink.com

info@cellink.com

5.

Crosslinking

MATERIALCrosslinking Agent
Cell culture medium
DESCRIPTIONCELLINK RGD is 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 rinseconstructs with basal culture media once.
Note: 30 seconds is recommended for 10 µL droplets while 10 minutes might be required for dense 1 cm3blocks. In addition, optimize the crosslinking depending on the cell type.
•6.

Incubation

MATERIAL
Cell culture medium
DESCRIPTION••

After crosslinking and washing, add the desired medium to the constructs 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 application. Replace medium regularly.

www.cellink.com

info@cellink.com