Ref No: REP- IKA325000503
Date: 03-JAN-2023
Author: AB, VK. Version: 2DILUTION 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 on
how to dilute the Alginate 5% to your desiredconcentration using the Reconstitution Agent M. The
obtained alginate hydrogel can be used on its own oras an additive in other bioink formulations. The
included Crosslinking Agent ensures stable ioniccrosslinking of alginate constructs for 3D cellculturing.

••••••••

Alginate 5%*
Reconstitution Agent M* or an alternative bufferof choice
Syringes with Luer lock connections
Female/female Luer lock adaptor*
Cartridge, 3cc
Bioprinting nozzles
BIO X*, BIO X6* or INKREDIBLE series* 3D
bioprinterCrosslink Agent* (included with the bioinkpurchase)*The product can be purchased in the CELLINKshop at www.cellink.com/shop.

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Protocol
All alginate concentrations are calculated as weight of alginate per total weight of lginate and Reconstitution
Agent M (w/w).

1.

Defining desired concentrations

DESCRIPTION••

Record the desired final concentration of alginate hydrogel (cF).
Record the desired final volume of alginate hydrogel to prepare (V F).

See Figure 1 for difference in viscosity of alginate hydrogels at different concentrations.

2.

Calculations

DESCRIPTION••

Calculate the volume of Alginate 5% to be used: 𝑉𝐴𝑙𝑔5% =𝑉𝐹 ·𝑐𝐹
5%

See Table 1 for suggested cF.
Calculate the volume of Reconstitution Agent M, VR, to be used. It is a specially designed buffer thatmaintains a physiologic pH in the final alginate bioink. It also has a low concentration of ions to prevent
premature crosslinking: 𝑉𝑅 = 𝑉𝐹 − 𝑉𝐴𝑙𝑔5%

Table 1. Suggested concentrations and the corresponding volume of Alginate 5% and Reconstitution Agent M
used for the preparation of 5 mL alginate hydrogel.
Final concentration of alginate, cF (%)

Volume of Alginate 5%, VAlg5% (mL)

Volume of Reconstitution Agent M, VR (mL)

1143323.

Preparing an alginate bioink

MATERIAL
Reconstitution Agent M
Alginate 5%
Syringes with Luer lock connections
Female/female Luer lock adaptor

DESCRIPTION•••

Transfer VR of Reconstitution Agent M into a sterile syringe that can accommodate minimum VF.
Transfer VAlg5% of Alginate 5% into another sterile syringe.
Connect the two syringes using a Luer lock adaptor, make sure there are no air bubbles present. Mix
the two solutions by passing them back and forth between the syringes until homogenized.

Note: if air bubbles are introduced into the mixture, centrifuge at 1000 x g for 1 min to remove them.
Note: If not using the alginate bioink right away, store at 4-8°C.

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4.

Bioprinting

MATERIAL
Alginate bioink
Cartridge, 3cc
Bioprinting nozzles
BIO X, BIO X6 or INKREDIBLE+Crosslinking AgentDESCRIPTION••

Transfer the alginate bioink to a cartridge and cap with a bioprinting nozzle of choice. See theBioprinting Protocol Alginate 5% for a step-by-step instruction of bioprinting using an alginate bioink.
Crosslink the bioprinted structures using the Crosslinking Agent for 30 seconds to 5 minutes dependingon the size of the constructs.

Figure 1. Viscosity of reconstituted alginate at various concentrations over a shear rate range of 0.01 to 100 s -1,
25°C.

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