Ref No: PRP-VL-900000
Date: 09-JAN-2023
Author: MG. Version: 2

PREPARATION PROTOCOLCELLINK Glucomannan
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 forusing CELLINK Glucomannan as a thickener ofhydrogels to increase their printability. CELLINK
Glucomannan is a sterile powder that may bedissolved in water, a buffer solution or a hydrogel.
This document includes dissolution of the thickener
in a liquid or very low viscosity solution as well as
mixing of the dissolved thickener (glucomannan gel)with a hydrogel for thickening effect.
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CELLINK Glucomannan*
Reconstitution liquid (e.g. water, PBS, HBSS,
cell culture medium etc.)
Tubes (1-50 mL)
Female/female Luer lock adaptor*
Syringes with Luer lock connections
Positive displacement pipette + pipette tips
(optional)
Hydrogel to be thickened
Cells + cell culture medium
Cartridge, 3 cc*
CELLMIXER* (optional)
BIO X* or INKREDIBLE series* 3D bioprinterBioprinting nozzles* or needles*
Well plate or Petri dish**The product can be purchased in the CELLINKshop at www.cellink.com/shop/.

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Protocol for preparing a glucomannan gel

1.

Calculations

DESCRIPTION••••

Record the desired final concentration of glucomannan (cG).
Record the desired volume of gel to prepare (VL).
See Figure 1 for difference in viscosity of glucomannan gels of different concentrations.
Calculate the amount of glucomannan to be used: 𝑚𝐺 = 10 · 𝑉𝐿 · 𝑐𝐺 . See Table 1 with calculations forsuggested cG.

Note: This equation gives a final concentration in weight/volume. However, depending on the concentrationof thickener the mixture may swell resulting in a slightly increased final volume.

Figure 1. Viscosity of CELLINK Glucomannan dissolved in PBS at different concentrations over a shear rate
range of 0.002 to 500 s-1, 25°C.
Table 2. Suggested concentrations and the corresponding amount of glucomannan powder used for thepreparation of 5 mL glucomannan gel.
Concentration of , cG (%)
0.51232.

Volume of prepared gel, VL (mL)
5555

Mass of glucomannan, mG (mg)2550100150

Heating up the reconstitution liquidMATERIAL
Reconstitution liquid
Tube

DESCRIPTION••

Transfer VL of the reconstitution liquid to a sterile tube or container of your choice.
To speed up the dissolution of glucomannan, heat up your reconstitution liquid to ~50°C using a waterbath, laboratory oven or similar. If your liquid cannot withstand heating, this part can be skipped.

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3.

Weighing up glucomannanMATERIALTube
CELLINK Glucomannan

DESCRIPTION•

4.

Into a tube, weigh up mG of glucomannan powder.

Dissolving glucomannan

MATERIAL
Reconstitution liquid
CELLINK Glucomannan

DESCRIPTION••••

Into the tube with reconstitution liquid, add the glucomannan powder. To reduce the formation ofclumps, add the glucomannan in increments and mix with a spatula.
Vortex the mixture at high speed until dissolved. If clumps form, crush them with a spatula.
Let the glucomannan gel rest for a minimum of two hours for it to reach its final viscosity.
Store at 4-25°C.

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Protocol for thickening a hydrogel using glucomannan
This approach may be preferred when the hydrogel is based on a high molecular weight biopolymer, is temperaturesensitive, has a high solid content or similar.

1.

Calculating desired final propertiesDESCRIPTION•••

Record the desired volume of hydrogel and glucomannan gel mixture (V2).
Record the desired final concentration of glucomannan in the mixture (c 2).
Record the concentration of glucomannan gel (c1). Follow the protocol above to prepare a glucomannangel of desired concentration.

•Calculate the volume of glucomannan gel (V1) to be used: 𝑉1 = 2 2•

Calculate the volume of hydrogel (VH) to be mixed with the glucomannan gel: 𝑉𝐻 = 𝑉2 − 𝑉1𝑉 ·𝑐𝑐1

2.

Mixing glucomannan gel and hydrogel

MATERIAL
Glucomannan gel
Hydrogel
Tube
Female/female Luer lock adaptor
Syringes with Luer lock connections
Positive displacement pipette + pipette tips (optional)

DESCRIPTION•••

Transfer the calculated volume of glucomannan gel and hydrogel into a sterile tube or the container ofyour choice. Mix with a sterile spatula.
Vortex the gel mixture at high speed until it appears homogenous.
If vortexing is not enough for mixing, use two syringes instead. Transfer the mixture to a syringe,
connect the syringe with another syringe of the same size using a female/female Luer lock adaptor. Mixby pushing the gel back and forth between the two syringes. This method may introduce air bubbles thatcan be removed by centrifuging the syringe for 1-2 min at 300 g.

Note: Transferring viscous gels may be difficult using a normal pipette. If available, use a positive
displacement pipette instead.

3.

Mixing with cells

MATERIAL
3 mL syringes with Luer lock connections
Female/female Luer lock adaptor
Hydrogel
Cell suspension
Cartridge, 3cc
CELLMIXER (optional)

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DESCRIPTION•••At this point, mix ten parts of hydrogel with one part of cell suspension, taking care not to introduce air
bubbles to the mixture. For detailed instructions see the Mixing cells with bioink Protocol.
If preparing for quantities < 2 mL of hydrogel, it is recommended to connect two 3 mL Luer locksyringes, one with the bioink and the other with the cell suspension and gently mix back and forth
between the syringes 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 hydrogel 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: To avoid introducing air when connecting the syringes, carefully pre-fill the Luer lock adaptor withhydrogel before attaching it to the syringe with the cell suspension.

4.

PrintingMATERIALBIO X, BIO X6 or INKREDIBLE series bioprinterBioprinting nozzles or needles
Well plate or Petri dishDESCRIPTION•••

Cap the cartridge with a bioprinting nozzle or a needle.
Place the cartridge in the printhead of the 3D bioprinter. Place the well plate or Petri dish on the printbed.
3D bioprint the hydrogel mixture according to the application.

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