Ref No: FBPR-KTB000000201
Date: 03-JAN-2023
Author: FS, VK, MSL. Version: 2

FRESH BIOPRINTING PROTOCOL

Alginate
This is a suggested procedure, please adjust it 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 complex 3D structures with low
viscosity alginate using FRESH printing method. Itcovers the steps of pre-print procedures, printing,andpost-printcrosslinking.
Changingtheparameters in the protocol might change printing
conditions such as pressure and speed. Thisprotocol was optimized for the Temperaturecontrolled printhead using the BIO X.

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Alginate Lyophilizate (100 mg vial)*
Reconstitution Agent M* or other buffer
Magnetic stir bar
Crosslinking Agent (50 mM CaCl2)*
LifeSupportTM*
3 mL syringes with Luer lock connections
Well plate or Petri dish*
BIO X*, BIO X6* 3D bioprinterCells in suspension and cell culture media
Female/female Luer lock adaptors*
Cartridge, 3cc*
22G Conical bioprinting needle (1-inch length)**The product can be purchased in the CELLINKshop at www.cellink.com/shop.

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Protocol
This protocol is adjusted for printing scaffolds at a final alginate concentration close to 2% (w/v) prepared from
Alginate Lyophilizate (CELLINK). For other alginate materials and concentrations, recalculations need to be madebut the same protocol can be followed. Once compacted, LifeSupport™ should be kept in a fridge at 4°C and usedwithin 12 hours. The ambient temperature should not exceed 23°C during handling or printing.

1.

Preparing Alginate 2% (w/v)

MATERIAL
Alginate Lyophilizate
Reconstitution Agent M
Magnetic stir bar
3 mL syringes with Luer lock connections

DESCRIPTION▪ Take Alginate Lyophilizate from storage and let it reach room temperature.
▪ Add 5 mL of a reconstitution agent to the Alginate Lyophilizate vial.
Note: Reconstitution Agent M is a specially designed buffer that maintains a physiologic pH in the final alginate
bioink. It also has a low concentration of ions to prevent premature crosslinking.
▪ Add a sterile stir bar to the container.
▪ Stir the mixture at room temperature for ~60 minutes, or until dissolved.
▪ Transfer the dissolved alginate to a syringe with Luer lock connection.

2.

Preparing LifeSupportTM bath with 0.1% (w/v) CaCl2MATERIAL
Crosslinking Agent
Reconstitution Agent M
LifeSupportTM
Well plate or Petri dish

DESCRIPTION▪ Prepare 40 mL of 0.1% (w/v) CaCl2 solution by mixing 7.2 mL of Crosslinking Agent with 32.8 mL ofReconstitution Agent M. Put the solution in a fridge (4°C) for cooling.
▪ Add 40 mL of cold CaCl2 solution to LifeSupportTM tube (2 g of sterile powder).
Note: 2 g of sterile LifeSupportTM powder corresponding to 15 mL of LifeSupportTM bath. For detailed directions,visit https://www.cellink.com/wp-content/uploads/2022/03/FluidformDirectionsforUseVersion6-rev-Sep-2021.pdf.
▪ Vortex for 1 minute.
▪ Put the tube into the fridge (4°C) for 15 minutes.
▪ Centrifuge for 5 minutes at 400 g.
▪ Gently pour off or aspirate the liquid supernatant.
▪ Grab the tube by the cap, hold it horizontally, and gently tap it against a palm 15 times.
▪ Shake the tube containing dislodged LifeSupport™ vigorously for 10 seconds. Shake along the length of thetube.
▪ Centrifuge for further 5 minutes at 400 g.

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▪ The LifeSupport™ should now be compacted at the bottom of the centrifuge tube. Gently pour off or aspirate
any remaining liquid supernatant to leave only the compacted LifeSupport™ in the bottom of the tube.
▪ Transfer the resulting LifeSupportTM bath with a sterile spatula into well plates or Petri dish and store it in afridge until use.

3.

Preparing for printing

MATERIAL
Alginate 2% (w/v)
BIO X or BIO X6 bioprinterCell suspension in syringe
Luer lock adaptors
Cartridge, 3cc
22G Conical bioprinting needle (1-inch length)

DESCRIPTION▪ Set the BIO X print bed at 10°C to guarantee LifeSupportTM bath stability.
▪ To prepare 1 mL of an alginate bioink for printing, mix 900 μL of Alginate 2% (w/v) solution with 100 μL ofcells suspension between syringes using a Luer lock adaptor until complete homogenization.
Note: To avoid an air gap and bubbles during mixing, carefully pre-fill the Luer lock adaptor with alginate before
attaching the syringe with the cell suspension.
▪ Load a cartridge with the alginate and cells mixture using a Luer lock adaptor. Place the cartridge in theprinthead and cap with the printing needle.

4.

Printing

MATERIALBIO X or BIO X6 bioprinterWell plate or Petri dish previous filled with LifeSupportTM bath
Cartridge with the alginate bioink

DESCRIPTION▪ Place the well plate or Petri dish previously filled with LifeSupport TM bath and place it on the print bed. Printconstructs using suggested parameters:•pressure at 18-20 kPa.

•speed at 3.5 mm/s.

Note: If printability is not as desired, adjust the pressure and/or speed to up/down to extrude more/less material
at different speeds.

5.

Incubation and crosslinking

MATERIAL
Cell culture medium

DESCRIPTION

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▪ After printing, incubate the constructs for 30 minutes at 37 °C (5% CO2 and 95% relative humidity) for bioink
crosslinking and LifeSupportTM melting.
Note: Large volumes may require longer times for the supporting bath to fully melt, though too long crosslinking
time can cause shrinkage of the constructs.
▪ Remove melted LifeSupportTM by replacing it with a warm cell medium to avoid handling the printed construct.
For example, if you printed into a 6-well plate, this can be done by carefully aspirating 2 mL of melted
LifeSupport™ out and adding 2 mL of warm cell media. Repeat this process until most of the support bathhas been replaced by the medium.

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