Ref No: BPR-IK-352102
Date: 07-FEB-2023
Author: EP,JH,VK. Version: 3

BIOPRINTING PROTOCOL

GelMA AThis 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 GelMA A bioink using the BIO X or BIO
X6, with and without cells. This document covers preprint mixing with cells, 3D bioprinting and post-print
processes such as ionic crosslinking andphotocuring. This protocol was optimized for GelMA
A with LAP at 0.25% concentration, undiluted as wellas with a 10+1 parts cell suspension dilution.
Changing the bioink to cell suspension ratio and thus
also the concentration of photoinitiator, will change
the photocrosslinking time. Refer to thePhotocrosslinking
Crosslinking
Optimization
Protocol to adjust and determine these numbers.
This protocol was optimized using the Temperaturecontrolled printhead with the BIO X system.

•••••
•
•••

www.cellink.com••GelMA A 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+* 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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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 with the BIO X or BIO X6 and the Temperature-controlled printhead as well as the cooled
print bed. While the bioink can be used with the INKREDIBLE+ system due to its ability to heat the bioink, secondary
steps are necessary to cool the printed structure to pre-gel it prior to crosslinking. Clogging may still occur due tolack of temperature control at the nozzle. Therefore, it is not recommended to use the bioink with the INKREDIBLE
system since the bioink will not perform as expected and resulting filament characteristics may be inconsistent. If
using the INKREDIBLE+ system, pre-heat a printhead to 24°C to achieve the same temperature maintenance as
the Temperature-controlled printhead. After deposition, the Petri dish or well plate should be placed on ice oranother cooled surface to thermally gel the construct after printing prior to photocrosslinking. First time users ofGelMA based bioinks are recommended to optimize the printing conditions without cells before proceeding tobioprint with cells. Perform the desired dilution using medium or PBS.

1.

Preparing the bioink

MATERIALGelMA A
3 mL syringes with Luer lock connections
Female/female Luer lock adaptor
Pipette tip or spatula

DESCRIPTION•Heat up the GeMA A cartridge at 37°C until it becomes liquid. The heating of the GelMA A can be
performed in a printhead or incubator and usually requires 30-60 min if the bioink is taken directly fromthe 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 GelMA A before re-attaching the cartridge.
Gently mix the bioink back and forth between the cartridge and syringe to homogenize the bioink. If notusing 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.
Note: If there are bubbles in the bioink, make a quick centrifugation for 30 s at 600 x g.
•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

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Pre-warmed GelMA ACell suspension
UV shielding 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 GelMA A, 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 GelMA A 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 withGelMA A before attaching it to the syringe with the cell suspension.

3.

Preparation for printing

MATERIAL
GelMA A mixed with cells (if applicable) in UV shielding cartridge
Temperature-controlled printhead (optional)
Conical bioprinting nozzles, 22-27G

DESCRIPTION•••

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 GelMA A in the printhead. Connect thecartridge 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 GelMA A, 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 increasestepwise.

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4.

Printing

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

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 300ms preflow delay, the suggested pressure range is between 22-32
kPa without cells and 18-28 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

MATERIAL
Crosslinking AgentAND/OR
405/365 nm LED modules for photocuring
Cell culture medium

DESCRIPTION•GelMA A can be crosslinking using the CaCl2-containing Crosslinking Agent or with photocrosslinking.
GelMA A with LAP can be crosslinked with either the 405 or 365 nm LED module. If using both, begin
with photocrosslinking. It is recommended to use either photocrosslinking alone for softer constructs orboth crosslinking methods which will generate more robust constructs. Optimize the crosslinking
according to application.

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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 crosslinking times. Ensure that the bioprinted
GelMA A construct is thermally gelled after printing by cooling the print bed. If photocrosslinking during
bioprinting, set the crosslinking parameters appropriately in the printhead setup page for the BIO X orBIO X6.

Note: To verify that the photocrosslinking is sufficient, add 37°C medium to one printed well and observe thatit doesn’t dissolve.
•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 seconds to crosslink the construct**. Distance from photocuring module to construct set
at 5 cm using the BIO X or BIO X6 photocuring modules. If using the INKREDIBLE+ photocuring modules, the timerequired can possibly be decreased. For crosslinking with other parameters, see Photocrosslinking Optimization
Protocol. This table was generated using GelMA A with mesenchymal stem cells. Don’t exceed 120 seconds ofexposure 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 light respectively, every second or
fourth layer.
1 mm construct thickness
3 mm construct thickness

365 nm, LAP 0.25%
5 seconds
15 seconds

405 nm, LAP 0.25%
10 seconds
30 seconds**Note this is only a recommended reference of starting times. The actual time needed for crosslinking will vary depending on
the size and temperature of the constructs as well as the intensity of the photocuring module and the distance to the construct.

6.

Incubation

MATERIAL
Cell culture medium

DESCRIPTION••

After crosslinking, add the desired medium to the constructs and place in an incubator.
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. Replace medium regularly.

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