Ref No: BPR-IK-3CT102
Date 07-FEB-2023
Author: PT, VK, ELG. Version: 3

BIOPRINTING PROTOCOLBIO CONDUCTINKThis 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 Bio Conductink using the BIO X or BIO
X6. This document covers pre-print mixing with cells,
3D bioprinting and post-print processes such as
photocuring. This protocol was optimized for BioConductink containing 0.25% LAP as aphotoinitiator. Changing the bioink to cell suspension
ratio and thus the concentration of photoinitiator, willchange the photocrosslinking time. Refer to thePhotocrosslinking Optimization Protocol to adjust
and determine these numbers. This protocol wasoptimized
using
theTemperature-controlled
printhead with the BIO X.

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Bio Conductink*
Cells* + cell culture medium*
Female/female Luer lock adaptor*
3 mL syringes with Luer lock connections
CELLMIXER* (optional)
UV shielding cartridges, 3 cc*
Conical bioprinting nozzles, 22-27G
recommended*
Temperature-controlled printhead*
BIO X* or BIO X6* 3D bioprinterWell plate or Petri dish**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.

ProtocolBio Conductink has been optimized for the BIO X system equipped with the Temperature-controlled printhead, athermal nozzle cover and a cooled print bed. While the bioink can be used with the INKREDIBLE+ system due toits ability to heat the bioink, secondary steps are necessary to cool the printed structure to pre-gel it prior tophotocrosslinking. In addition, clogging may still occur due to lack 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 expectedand resulting filament characteristics may be inconsistent. First time users of GelMA based bioinks arerecommended to optimize the printing conditions without cells before proceeding to bioprint with cells. Perform thedesired dilution using medium or PBS.

1.

Preparing the bioink

MATERIAL
Bio Conductink
Temperature-controlled printhead

DESCRIPTION•••

2.

Heat up Bio Conductink in the cartridge at 37°C until it is liquid. The heating of the Bio Conductink canbe performed in a printhead or incubator and usually requires 30-60 min if the bioink is taken directlyfrom the fridge.
Set the Temperature-controlled printhead to 25°C.
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 Bio Conductink
Cell suspension
UV shielding cartridge, 3cc
CELLMIXER (optional)

DESCRIPTION•••At this point, mix ten parts of bioink with one part of 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 Bio Conductink, 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 Bio Conductink to the 12 mL syringe (PART 2) using a female/female Luer lockadaptor.
•Clip both syringes to the Dispensing unit (PART 3).

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••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 Bio
Conductink before attaching it to the syringe with the cell suspension.
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 when liquid.

3.

Preparation for printing

MATERIAL
Bio Conductink 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 25°C for 10 minutes. If not using theTemperature-controlled printhead, place the cartridge on counter for 5-10 minutes to reachapproximately 25°C.
If the cartridge has cooled down below 24°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 Bio Conductink 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 Bio Conductink, the recommended printhead temperature for the highest printing
fidelity is 25°C, though the bioink can be dispensed up to 32°C. Below 24°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.

4.

PrintingMATERIALBIO X or BIO X6
Well 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. The
printing pressure is inversely proportional to a nozzle diameter and printing speed.

Example: If printing continuous filaments with a Temperature-controlled printhead set to 25°C, a 25G nozzle,
a printing speed of 5 mm/s and with 300 ms pre-flow delay, the suggested pressure range is between 23-33
kPa without cell suspension dilution and 18-28 kPa if diluted with cell suspension.

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•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.
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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 25°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 25°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
405/365 nm LED modules for photocuring

DESCRIPTION•••Bio Conductink with LAP can be crosslinked with photoinitiation using either the 405 or 365 nmphotocuring module. The photocrosslinking time required is usually in the range of 15-30 s for aconstruct thickness below 3 mm. More time might be required depending on the construct thickness anddesired final stiffness. Ensure that the bioprinted Bio Conductink construct is thermally gelled after
printing by letting the construct sit on the cooled print bed for 30 s before starting photocrosslinking.
If photocrosslinking during bioprinting, set the crosslinking parameters appropriately in the printhead
setup page for the BIO X or BIO X6. To achieve the better structural integrity when printing thicker
constructs, it is recommended to apply 3 or 5 seconds photocrosslinking with 365 or 405 nm lightrespectively, every second or fourth layer.
Let the structure sit for 3-5 min to allow crosslinking after the light source is turned off.

Note: It is recommended to use the 405 nm LED module instead of 365 nm if possible. Overexposure might
damage the cells.
Note: To verify that the photocrosslinking is sufficient, add 37°C medium to one printed well and observe thatit doesn’t dissolve.

6.

Incubation

MATERIAL
Cell culture mediumDESCRIPTION••

After photocrosslinking, add the desired medium to the construct and place in the 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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