Ref No: REP-VLB00000
Date: 03-JAN-2023
Author: AB, MG. Version: 4

RECONSTITUTION PROTOCOL

Alginate Lyophilizate
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 reconstitute Alginate Lyophilizate to yourdesired concentration using Reconstitution Agent M.
The obtained alginate hydrogel can be used for 3D
bioprinting and cell culturing, casting applications
and mixing with other biomaterials. Alginatehydrogels can be crosslinked with calcium containing
solutions to ensure stable constructs for cell culture.

•••••
•
•

Alginate Lyophilizate (2 x 100 mg), sterile*
Reconstitution Agent M* or an alternative bufferof choice
Magnetic stir bar
Cartridges, 3cc
Bioprinting nozzle
BIO X*, BIO X6* or INKREDIBLE series* 3D
bioprinterCrosslinking Agent**The product can be purchased in the CELLINKshop at www.cellink.com/shop.

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Protocol
This protocol describes reconstitution of 100 mg of Alginate Lyophilizate to obtain bioinks of different
concentrations. All alginate concentrations are calculated as weight of alginate per total weight of Alginate andReconstitution Agent M (w/w).

1.

Preparing Alginate Lyophilizate

MATERIALAlginate Lyophilizate

DESCRIPTION•

2.

Take Alginate Lyophilizate from storage and let it reach room temperature.

Preparing Reconstitution Agent M

MATERIAL
Reconstitution Agent M

DESCRIPTION•Prepare 12 mL of Reconstitution Agent M.

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.

3.

Preparing alginate bioink

MATERIAL
Alginate Lyophilizate
Reconstitution agent M
Stir bar

DESCRIPTION••••

Add the desired amount of Reconstitution Agent M to the Alginate Lyophilizate vial, see Table 1 forsuggested alginate concentrations and Figure 1 for their viscosity.
Add a sterile stir bar to the container.
Stir the mixture at room temperature for ~60 minutes, or until dissolved.
Double check that pH is in the 6.5-7.4 range. If needed, balance with small volumes of NaOH or HCl. If
not using right away, store at 4-8°C if needed.

Note: Adding additional liquids to adjust the pH dilutes your bioink.

Table 1. Suggestions of final alginate concentrations for reconstitution of 100 mg Alginate.
Concentration of Alginate (w/w)
1%
3%
5%

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Volume of Reconstitution Agent M needed
9.9 mL
3.23 mL
1.9 mL

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4.

Bioprinting and crosslinking

MATERIAL
Alginate bioink
Cartridges, 3cc
Bioprinting nozzle
BIO X, BIO X6 or INKREDIBLE bioprinterCrosslinking AgentDESCRIPTION•

Use the alginate bioink as it is or mix it with other components of choice, see Mixing cells with bioink
Protocol for instructions on how to mix the bioink with cells.
•Transfer the alginate bioink into a cartridge and cap with a bioprinting nozzle of choice.
•Bioprint structures according to application. See the Bioprinting Protocol Alginate 5% for an example ofprinting alginate hydrogel with cells.
•Alginate bioinks can be crosslinked by submerging the bioprinted constructs in Crosslinking Agent for 30
s to 5 min depending on construct size.
Note: 30 seconds is recommended for small droplets while 5 minutes for dense >1 cm 3 blocks.

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

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