Ref No: BPR- IKA325000503
Date: 02-JAN-2023
Author: EP,JH,VK. Version: 3BIOPRINTING PROTOCOL

Alginate 5%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 Alginate 5% using the INKREDIBLE,
INKREDIBLE+, BIO X or BIO X6. The protocol
covers the steps of pre-print mixing with cells, 3D
bioprinting and post-print processes such as ioniccrosslinking. It was optimized for Alginate 5% diluted
with a cell suspension at a 10+1 volume ratio.
Changing the parameters in the protocol mightchange the crosslinking time required. This protocol
was optimized for the pneumatic printhead on theBIO X and BIO X6. For printing complex multilayered structures, it is recommended to mix youralginate hydrogel with a thickener such as
Nanofibrillated Cellulose, Xanthan Gum orGlucomannan also available from CELLINK.

•••••••••••

Alginate 5%Cells* + cell culture medium*
Female/female Luer lock adaptors*
3 mL syringes with Luer lock connections
Positive displacement pipette + pipette tips
(optional)
CELLMIXER* (optional)
Cartridges, 3cc*
Bioprinting nozzles or needles, 22G-27G
recommended*BIO X*, BIO X6* or INKREDIBLE series* 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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Protocol
This protocol can be performed with printheads and print bed at room temperature, i.e. 20-25°C.

1.

Preparing the bioink

MATERIAL
Alginate 5%

DESCRIPTION•••2.

If applicable, dilute Alginate 5% to the desired concentration, see Dilution Protocol Alginate 5%.
Warm up the Alginate solution to 37°C to make it softer and thus easier to mix with cells (refer to Figure1).
If not printing with cells, transfer the alginate to a cartridge and 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 Alginate solution
Cell suspension
Cartridge, 3cc
CELLMIXER (optional)
Positive displacement pipette + pipette tips (optional)

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 Alginate, 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 thesyringes 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.

Note: Transferring viscous Alginate solutions may be facilitated by using a positive displacement pipette.
Note: To avoid introducing air when connecting the syringes, carefully pre-fill the Luer lock adaptor with Alginate
before attaching it to the syringe with the cell suspension.

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3.

Preparation for printing

MATERIAL
Alginate mixed with cells (if applicable) in cartridge
Bioprinting nozzles or needles, 22-27G

DESCRIPTION••

If applicable, set the desired temperature on the Temperature-controlled printhead or pneumaticprinthead.
Cap the cartridge with a printing nozzle or needle of choice and place in printhead. Connect thecartridge to the air adapter.

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 dish

DESCRIPTION•

Bioprint structures using suggested parameters (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: Using the Syringe Pump printhead in combination with the BIO X allows for better control over extrusion
rates.

Table 1. Recommended minimal extrusion pressure** (±2 kPa) used for printing continuous filaments at 20-25°C
diluted/undiluted bioink. ‘Diluted’ assumes a mixture of one part of cell suspension and ten parts of bioink. For smaller
dilutions, the pressure needs to be increased towards the pressure used for undiluted bioink.
Printing speed (mm/s) →Nozzle size (G) ↓52292592710101616161113131520212213161620232530161919293136**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 printhead temperature of 24°C and with a 10+1 bioink dilutionwith PBS.

5.

Crosslinking

MATERIALCrosslinking Agent
Cell culture medium

DESCRIPTION
Alginate hydrogels can be crosslinked with ions using the CaCl2 containing Crosslinking Agent.

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•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 the 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.

6.

Incubation

MATERIAL
Cell culture medium

DESCRIPTION••

After crosslinking and washing, add the desired medium to the constructs and place them in anincubator.
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.

Figure 1. Viscosity behavior of Alginate 5% at different temperatures, over a shear rate range of 0.01 to 100 s -1.

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