Ref No: BPR-IK-B118L3
Date: 07-FEB-2023
Author: EZ,EP,JH,VK. Version: 4

BIOPRINTING PROTOCOLGelXA CARTILAGE
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 GelXA CARTILAGE bioink using theINKREDIBLE, INKREDIBLE+, BIO X or BIO X6, withand without cells. This document covers pre-print
mixing with cells, 3D bioprinting and post-printprocesses such as such as ionic crosslinking andphotocuring. This protocol was optimized for GelXACARTILAGE with LAP 0.25% undiluted as well as a10+1 cell suspension dilution. Changing the bioink tocell suspension ratio and thus also the concentrationof photoinitiator, will change the photocrosslinking
time. Refer the Photocrosslinking Optimization
Protocol to adjust and determine these numbers.
This protocol was optimized using the Temperaturecontrolled printhead using the BIO X.

•••••••••

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GelXA CARTILAGE bioink*Cells* + cell culture medium*3 mL syringes with Luer lock connections
Female/female Luer lock adaptor*
CELLMIXER* (optional)
UV shielding cartridges, 3cc*
Conical bioprinting nozzles, 22-27G*
Temperature-controlled printhead (optional)
BIO X*, BIO X6* or INKREDIBLE series 3D
bioprinter*
Well 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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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.

ProtocolThis protocol works best using the BIO X or BIO X6 with the cooled print bed at 15°C and the Temperaturecontrolled printhead at 24°C. The GelXA CARTILAGE can also be extruded using the BIO X/BIO X6 pneumaticprintheads or the INKREDIBLE series, but with decreased shape fidelity if ambient temperature exceeds 25°C andthe printhead heats up. If using the INKREDIBLE series, the printing substrates such as Petri dishes or well plates
should be placed on ice or another cooled surface to thermally gel the construct after printing prior tophotocrosslinking. First time users of GelMA based bioinks are recommended to optimize the printing conditions
without cells before proceeding to bioprint with cells. Perform the desired dilution using medium or PBS.

1.

Preparing the bioink

MATERIAL
GelXA CARTILAGE
3 mL syringe with Luer lock connections
Female/female Luer lock adaptor
Pipette tip or spatula

DESCRIPTION•Heat up the GelXA CARTILAGE cartridge at 37°C until it becomes liquid. The heating of the GelXACARTILAGE can be performed in a printhead or incubator and usually requires 30-60 min if the bioink istaken directly from the fridge.
Note: Keep the cartridge horizontal when tip and end cap are removed. This is to prevent air from entering
the cartridge or bioink from dripping out.
•To make sure that the bioink is homogeneous after heating, connect the cartridge to a 3 mL syringe
using a Luer lock connector and remove the end cap. Push approximately half of the bioink from thecartridge into the syringe by gently pushing the cartridge piston using a pipette tip or small spatula while
simultaneously pulling the syringe plunger. To remove any air bubble derived from the dead volume inthe syringe, separate the syringe and cartridge maintaining the Luer lock on the syringe. Hold thesyringe with the tip facing upwards and gently tap the syringe to move air bubbles towards the tip.
Carefully extrude air and pre-fill the Luer lock adaptor with GelXA CARTILAGE before re-attaching thecartridge. Gently mix the bioink back and forth between the cartridge and syringe to homogenize thebioink. If not using the entire 3 mL of the bioink in the cartridge, keep the rest of the bioink in the optimal
storage conditions. Prolonged and repeated heating could negatively affect the photoinitiator stability.
•If not printing with cells move directly to Step 3.

2.

Mixing the bioink with cells

MATERIAL
3 mL syringes with Luer lock connections
Female/female Luer lock adaptor
Pre-warmed GelXA CARTILAGE
Cell suspension
UV shielding cartridge, 3cc
CELLMIXER (optional)

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DESCRIPTION•••At this point, mix ten parts of bioink with one part of cell suspension, taking care not to introduce airbubbles to the mixture. For detailed instructions see the Mixing cells with bioink Protocol.
If preparing for quantities < 2 mL of GelXA CARTILAGE, it is recommended to connect two 3 mL Luer
lock syringes, 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 GelXA CARTILAGE to the 12 mL syringe (PART 2) using a female/female Luer lockadaptor.
Clip both syringes to the dispensing unit (PART 3).
Connect the two syringes to the mixing unit (PART 4), then connect the Empty cartridge(PART 5) 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
GelXA CARTILAGE before attaching it to the syringe with the cell suspension.

3.

Preparation for printing

MATERIAL
GelXA CARTILAGE mixed with cells (if applicable) in UV shielding cartridge
Temperature-controlled printhead (optional)
Conical bioprinting nozzles, 22-27GDESCRIPTION•••

If the cartridge is just taken from the heat or if the cartridge still feels warm after mixing in the cells, place
it in the pre-heated Temperature-controlled printhead at 24°C for 10 minutes. If not using theTemperature-controlled printhead, place the cartridge on counter for 5-10 minutes to reachapproximately 24°C.
If the cartridge has cooled down below 23°C, re-heat the cartridge at 37°C for 5 min to reset, thenrestart equilibration in printhead.
Cap the cartridge with a bioprinting nozzle and place the GelXA CARTILAGE in the printhead. Connect
the cartridge to the air pressure adapter. If using the BIO X or BIO X6, pre-cool the print bed to 15°C.

Note: When printing with GelXA CARTILAGE, the recommended printhead temperature for the highest
printing fidelity is 24°C, though the bioink can be dispensed up to 32°C. Below 23°C the bioink can become
too viscous resulting in chunky filaments and too high extrusion pressures needed.
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 dishwww.cellink.com

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DESCRIPTION•Calibrate the nozzle to the well plate or Petri dish surface. Test the flow of the bioink first after calibrationand start with a low pressure and increase stepwise. Bioprint structures into the well plate or Petri dish.
If printability is not as desired, adjust the pressure up/down by 1 kPa to extrude more/less material.
Example: If printing continuous filaments with a Temperature-controlled printhead set to 24°C, a 25G nozzle,
a printing speed of 5 mm/s and with 300 ms pre-flow delay, the suggested pressure range is between 26-36
kPa without cell suspension dilution and 21-31 kPa if diluted with cell suspension.
•If proper viscosity and printability is not achieved by extending temperature equilibration time or tuningpressure:•Too low viscosity (wide filaments despite using low pressure): decrease the printheadtemperature 0.5-1°C to increase the viscosity and equilibrate an additional couple of minutes.
•

Too high viscosity (chunky filaments and high pressure required): increase the printheadtemperature 0.5-1°C to decrease the viscosity and equilibrate an additional couple of minutes.
If unsuccessful, the bioink might have over-gelled. In this case, re-heat the cartridge at 37°C for
5 min to reset and choose a 1°C higher printhead temperature than before. Let the bioink reachthe new temperature before starting to print again.

•During print sessions longer than 20 min at 24°C the bioink can become too viscous due to continued
gelling resulting in chunky filaments and too high extrusion pressures needed. To avoid this, a 0.5°C
increase in printhead temperature after 20 min printing can extend the time the bioink remains at goodprintability.
•If the regular pneumatic printheads are used for a long period, they might heat up above the desired
printing temperature. Then the bioink also heats up which decreases its viscosity, observed as extrusionof very thick filaments even at low pressures. Remove the cartridge from the printhead and allow to cool
down to 24°C. In addition, remove the printheads from the BIO X/BIO X6 to let them cool down before
continuing to print.
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.

5.

Crosslinking

MATERIALCrosslinking Agent
AND/OR
405/365 nm LED modules for photocuring
Cell culture medium

DESCRIPTION•GelXA CARTILAGE can be photocrosslinked using the 405 or 365 nm LED modules or ionicallycrosslinked using the CaCl2-containing Crosslinking Agent. If using both, begin with photocrosslinking. It
is recommended to use either photocrosslinking alone for softer constructs or both crosslinking methods
which will generate more robust constructs. Optimize the crosslinking according to application.

Note: It is recommended to use the 405 nm LED module instead of 365 nm if possible. Overexposure might
damage the cells.
•Photocrosslinking: See Table 1 below for recommended photocrosslinking times. Ensure that thebioprinted GelXA CARTILAGE construct is thermally gelled after printing by cooling the print bed (ifusing the BIO X/BIO X6) or placing the printing substrates with the construct on ice for 10 s (if using theINKREDIBLE series). If photocrosslinking during bioprinting, set the crosslinking parameters
appropriately in the G-code for the INKREDIBLE series or the printhead setup page for the BIO X/BIOX6.

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

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•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 cm 3blocks. In addition, optimize the crosslinking depending on the cell type.
Table 1. Recommended time of the construct crosslinking**. Distance from each light module to construct was set
to 5 cm using the BIO X/BIO X6 photocuring modules. If using the INKREDIBLE series photocuring modules, thetime required can possibly be decreased. For crosslinking with other parameters, see Photocrosslinking
Optimization Protocol. This table was generated using GelXA CARTILAGE with mesenchymal stem cells Don’t
exceed 120 seconds of exposure time when printing with cells. To achieve the best structural integrity when printing
thicker constructs, it is recommended to apply 3 or 5 seconds photocrosslinking with 365 or 405 nm lightrespectively, every fourth layer. If the bioink is used above 25°C, the best results can be achieved whenphotocrosslinking every second layer.
Photocuring using 365 nm module
Photocuring using 405 nm module
Ionic crosslinking using Crosslinking Agent

Time for 25 µL droplet
5s
10 s
30 s

Time for 3 mm grid
15 s
20 s
5 min

**Note: This is only a recommended reference of crosslinking times to start with. The actual time needed for crosslinking will varydepending on the size and temperature of the constructs as well as the intensity of the photocuring module and the distance tothe construct.

6.

Incubation

MATERIAL
Cell culture medium

DESCRIPTION••

After crosslinking, add the desired medium to the constructs and place them in an 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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