Ref No: PRP-IK-T20000
Date: 09-JAN-2023
Author: MG. Version: 3

PREPARATION PROTOCOL

Nanofibrillated Cellulose
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 for
how to use CELLINKs Nanofibrillated Cellulose(NFC) as a thickener of hydrogels to increase theirprintability. NFC is a hydrogel of cellulose fibers
dispersed in water, with a dry content of 3%.

••••••••••••

Nanofibrillated Cellulose (NFC)*Hydrogel to be thickened
Tubes
Female/female Luer lock adaptor*
Syringes with Luer lock connections
Positive displacement pipette + pipette tips
(optional)
Cells + cell culture medium
Cartridges, 3cc*
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
Nanofibrillated cellulose can be mixed with protein solutions and biopolymers at neutral pH. Salt concentrations
above 0.4% may induce flocculation of the nanofibrils of pure NFC in water, causing a slightly less homogeneoushydrogel. No significant effect is normally seen on the flow viscosity on mixed formulations with NFC.

1.

Calculations

DESCRIPTION••••

Record the desired volume of hydrogel and NFC mixture (VF).
Record the desired final % concentration of NFC in the mixture (cF).
NFC has a concentration of 3%, thus cNFC=3.
See Figure 1 for difference in viscosity of NFC of different concentrations.

•Calculate the volume of NFC (VNFC) to be used: 𝑉𝑁𝐹𝐶 =•Calculate the volume of your hydrogel (VH) to be mixed with the NFC: 𝑉𝐻 = 𝑉𝐹 − 𝑉𝑁𝐹𝐶

𝑉𝐹 ·𝑐𝐹
𝑐𝑁𝐹𝐶

Figure 1. Viscosity of CELLINK Nanofibrillated Cellulose diluted with Reconstitution Agent M (sugar solution,available at CELLINK web store) to different concentrations over a shear rate range of 0.002 to 500 s-1,25°C.

2.

Mixing NFC and hydrogel

MATERIAL
Nanofibrillated Cellulose
Hydrogel
TubeFemale/female Luer lock adaptor
Syringes with Luer lock connections
Positive displacement pipette + pipette tips (optional)

DESCRIPTION••••

Transfer the calculated volume of NFC and hydrogel into a sterile tube, or the container of your choice.
Mix with a spatula to combine.
Vortex the gel mixture at high speed until it appears homogeneous and of uniform viscosity.
If vortexing is not enough, instead use two syringes to mix the gels. Transfer the mixture to a syringe,
connect the syringe with another syringe of the same size using a female/female Luer lock adaptor. Mix

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•by pushing the gel back and forth between the two syringes. This method may introduce air bubbles
which can be removed by centrifuging the syringe for 1-2 min at 200 – 500 g.
If not printing with cells, transfer the mixture to a cartridge and move directly to Step 5.

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)

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.

Bioprinting

MATERIAL
Bioprinting nozzles or needles
3D bioprinterWell plate or Petri dish.

DESCRIPTION••Cap the cartridge with a bioprinting nozzle or a needle. Place the cartridge in the printhead of the 3D
bioprinter and connect to the air adapter. Place the well plate or Petri dish on the print bed.
3D bioprint the hydrogel mixture according to desired application.

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