Ref No: BPR-IK-130065
Date: 02-JAN-2023
Author: EP, JH, IB, VK. Version: 5

BIOPRINTING PROTOCOLCELLINK SKIN
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 with the CELLINK SKIN 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 SKIN, undiluted as well as using a 10+1
cell suspension dilution. Changing the parameters inthe protocol might change the required crosslinking
time. This protocol was optimized using thepneumatic printhead on the BIO X.

•••••••••••CELLINK SKIN*
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)
Vial with 100 U thrombin (included with thebioink purchase)*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 SKIN
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 SKIN 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 SKIN 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 SKIN
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 SKIN, 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.

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

3.

Preparation for printing

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

Attach a nozzle to the cartridge and mount the room tempered CELLINK SKIN into the printhead.
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: Test the flow of the bioink after the calibration is performed and start with a low pressure and increasestepwise.

4.

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

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.
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.
Table 1. Recommended minimal extrusion pressure** (±2 kPa) used for printing continuous filaments at 21-25°C
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.
with cells/Printing speed (mm/s) →Nozzle size (G) ↓

522

925

1027

11

10
111215111113

15
1214
16

12
13
14

20
14
15
17

13
15
15

15
1720

**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 at 23°C and with a 10+1 bioink dilution
with cell suspension.

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5.

Crosslinking

MATERIAL
Crosslinking AgentThrombin
Cell culture medium
DESCRIPTION•CELLINK SKIN is crosslinked with ions using the CaCl2-containing Crosslinking Agent and thrombin.
•
Make a 100 U/mL thrombin stock: Add 1 mL of cell culture medium to the thrombin vial. If notusing all the bioink, aliquots of the thrombin stock can be stored at -70°C for 2 months.
The ionic and thrombin crosslinking can either be sequential or simultaneous.

•

Alternative 1, simultaneous ionic and thrombin crosslinking:•••Add 1 part of thrombin stock solution to a tube with 7 parts of cell culture medium and 2 parts
Crosslinking Agent to receive a final concentration of 10 U/mL thrombin and 10 mM CaCl2. Mix
gently by pipetting up and down 2-3 times. Submerge the samples in the thrombin containing
Crosslinking Agent and incubate overnight in standard culture conditions (37°C, 5% CO2 and95% relative humidity) or according to your application.

Alternative 2, sequential ionic and thrombin crosslinking:Ionic crosslinking: 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 cm3 blocks. In addition, optimize the crosslinking depending on the cell type.
••

6.

Thrombin: Add 1 part of thrombin stock solution to a tube with 9 parts of medium to receive a
10 U/mL final concentration of thrombin. Mix gently by pipetting up and down 2-3 times.
Submerge the samples in the thrombin containing medium and incubate overnight in standard
culture conditions (37°C, 5% CO2 and 95% relative humidity) or according to your application.

Incubation

MATERIAL
Cell culture medium
DESCRIPTION•The next day, switch the thrombin containing medium to regular cell culture medium and incubate instandard culture conditions or according to your application. Replace medium regularly.

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