Ref No: PPR- IKS400000503
Date: 02-JAN-2023
Author: VK, ELG, MG. Version: 2PRINTING PROTOCOL

Pluronics 40%
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 for3D printing of Pluronics 40% as a sacrificial ink, using
the INKREDIBLE Series, the BIO X or BIO X6. This
document covers pre-print operations, 3D printingand post-print processes such as removing thesacrificial ink through washing with cold PBS.
Diluting Pluronics 40% will reduce its printability.

••••••••Pluronics 40% *Cartridges, 3 cc*
Female/female Luer lock adaptor*
Conical bioprinting nozzles, 22-27G*
BIO X*, BIO X6* or INKREDIBLE-Series* 3D
bioprinterPetri dish* or well plate
Cold PBS (maximum 8°C)
Ice

*The product can be purchased in the CELLINKshop at www.cellink.com/shop.

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Protocol
Pluronics 40% has been optimized for the INKREDIBLE-series and BIO X/BIO X6 system equipped with apneumatic printhead.

1.

Ink preparation

MATERIAL
Pluronics 40%Cartridge, 3 cc
Female/female Luer lock adaptor
Conical bioprinting nozzles, 22-27G

DESCRIPTION•••

Connect the Pluronics syringe to a cartridge using a Luer lock adaptor and transfer desired amount into
the cartridge.
Heat Pluronics 40% to room temperature. At temperatures below 13°C it becomes very liquid and thus
loses its printability, see Figure 1 for temperature sweep of Pluronics 40%.
Cap the cartridge with a bioprinting nozzle and place the cartridge in the printhead.

Figure 1. Storage modulus of Pluronics 40% over a temperature range of 15 to 37°C.

2.

PrintingMATERIALBIO X, BIO X6 or INKREDIBLE series bioprinterPetri dish or well plate

DESCRIPTION••

Print support and/or sacrificial structures using the ink on to a Petri dish or well plate. If printability is not
as desired, adjust the pressure up/down by 1 kPa to extrude more/less material. The printing pressure isinversely proportional to the nozzle diameter and printing speed.
The relation between printing pressure and speed for 22–27G nozzles can be seen in Figure 2.

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Note: Test the flow of the bioink after the calibration is performed and start with a low pressure and increase
stepwise.
Example: If using a 22G nozzle and 10 mm/s printing speed, start at 70 kPa and adjust as needed.

Figure 2. Filament diameter of Pluronics 40%, 3D printed with various nozzles, pressure, and printing speed, at25°C.

3.

Washing

MATERIAL
Cold PBS
Ice

DESCRIPTION
••••

4.

Pluronics 40% becomes liquid at low temperatures and may thus be removed by washing with cold
PBS, or your liquid of choice. Prior to washing, make sure to crosslink any other bioinks printed to retain
their structure.
Cover the printed construct in cold PBS and remove by pipetting. Repeat until all Pluronics is removed.
If the Pluronics is embedded inside a construct, wash with cold PBS and use negative pressure toremove the ink.
Acellular and large constructs benefit from being placed on ice for faster liquification of the Pluronics.

Incubation

MATERIAL
Cell culture medium

DESCRIPTION••

After removing the Pluronics, add the desired medium to the constructs and place in 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.

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