Nanocelluloses are nano-scale materials with the advantages of non-toxicity, high surface area, super strength, tunable surface chemistry, biodegradability, biocompatibility, and promoting cell interaction and tissue development. Nanofibrillated cellulose (NFC), also known as cellulose nanofibers (CNF) or cellulose nanofibril, is one structural type of nanocellulosic materials. NFC has an abundance of hydroxyl groups on the fibril surfaces, which leads to strong hydrogen interactions between fibrils and with water molecules, giving viscoelasticity and shear thinning properties that are favorable for 3D bioprinting.
GrowInk™ Bioinks
Amerigo Scientific offers GrowInk™ bioinks that are animal-free, biocompatible, ready-to-use and can be mixed directly with cells for 3D bioprinting applications. GrowInk™ bioinks consist primarily of NFC and water, providing a fully defined and customizable matrix. The matrix can be customized by adding molecules, such as growth factors or adhesion proteins, according to the needs of specific cell culture.
Flexibility and Customizability
GrowInk™ bioinks mimic the in vivo environment that supports cell growth and differentiation. The stiffness of GrowInk™ can be tuned to match the requirements of specific cells. GrowInk™ is stable at room temperature and can be used with or without the cross-linking agent, depending on the application, but all enable cell printing layer-by-layer with high-precision cell positioning.
Compatibility
GrowInk™ bioinks have shear-thinning properties and are easy to print under force, and the gel retains shape and structure once printed. They are especially suitable for extrusion bioprinting with high printing accuracy. GrowInk™ have been used with many commercial and homemade bioprinters such as:
GrowInk™-ALG is an NFC based bioink with alginate that can be cross-linked.
GrowInk™ Specifications
Property
GrowInk ™-N
GrowInk ™-T
GrowInk ™-ALG
Composition
Nanofibrillar cellulose
Anionic nanofibrillar cellulose
Nanofibrillar cellulose plus alginate
Visual appearance
Opaque gel
Transparent gel
Opaque gel
Sterility
Sterile
Sterile
Sterile
Endotoxins
< 5 EU/ml
< 5 EU/ml
< 5 EU/ml
Cell viability (HepG2)
Tested
Tested
Tested
Viscosity (TA Hybrid Rheometer HR-1, frequency flow sweep)
1-55 000 Pas
1-45 000 Pas
1-30 000 Pas
pH
6.5 ± 0.5
6.0 ± 0.5
7.0 ± 0.5
Storage temperature
4-22 °C
4-22 °C
4-8 °C
3D Bioprinting of GrowInk™-N and GrowInk™-T with Poietis NGB-R™ bioprinter
The printability and cell compatibility of GrowInk™-N and GrowInk™-T with the NGB-R™ bioprinter employing a unique laser-assisted bioprinting technology were evaluated. GrowInk™-N and GrowInk™-T were easy to print by laser and were also successfully printed by pneumatic extrusion. Both bioinks remained stable over time. GrowInk™-N was slightly more viscous than GrowInk™-T, which resulted in slightly better structural stability of the printed objects.
Figure 1. GrowInk™-N (A) and GrowInk™-T (B) printed by laser. Small droplets (< 200 µm in diameter) were printed reproducibly with high precision.
Figure 2. GrowInk™-N (A and B) printed by extrusion.
Figure 3. GrowInk™-T (C and D) printed by extrusion.
Laser-assisted printing and extrusion printing of GrowInk™ were studied to prepare layered fibroblast/keratinocyte cell models to investigate the suitability of the bioinks for printing and tissue engineering applications. Fibroblasts were successfully laser-printed between the extrusion-printed GrowInk™-T layers, and the keratinocytes printed on top of GrowInk™-T showed correct morphology and good viability.
Figure 4. (A) Schematic representation of the 3D model printed with GrowInk™. Gel layers were printed by extrusion and the cells were printed by laser. (B) Histological characterization of the 3D printed model construct. Stained by Goldner, showing the structured morphology of keratinocytes on top of GrowInk™-T.
Tips for Using GrowInk™
GrowInk™ bioinks are particularly suited for extrusion bioprinting with high printing accuracy.
To minimize the risk of clogging, nozzles 20 to 32-gauge diameter are recommended.
Set the layer height equal to the inner nozzle diameter.
The following parameters are recommended for starting bioprinting tests: pressure 10–30 kPa and speed 10–20 mm/s.
Bioprinting with GrowInk™ at room temperature is recommended. Printing a cell-GrowInk™ mixture can be carried out at maximum 37°C.
When first working with GrowInk™, printing a regular rectilinear pattern is recommended.
When transferring Growink™ to alternative syringes, using a 10ml syringe is recommended.
If bioprinting has been paused for over 10 minutes, it is recommended to replace the nozzle, as GrowInk™ may have dried at the tip and clogged the nozzle.
Ensure that no bubbles are introduced when transferring the GrowInk™, as bubbles interfere with the printing performance.
Store GrowInk™ correctly and use within the expiration date. The expiration date of the GrowInk™ bioinks is normally 6 or 12 months from manufacture depending on the product. GrowInk™ should be stored as specified on the product label, normally 4-22°C (39–72°F) and protected from light for optimum performance. Once opened, it is recommended to store the product at 4-8°C (39-46°F) for up to 3 months. Ensure the cartridges are capped before storage to prevent drying.
Do not store GrowInk™ below 0°C (32°F). Freezing can cause destabilization of the product and render it unusable.
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Figure 4. (A) Schematic representation of the 3D model printed with GrowInk™. Gel layers were printed by extrusion and the cells were printed by laser. (B) Histological characterization of the 3D printed model construct. Stained by Goldner, showing the structured morphology of keratinocytes on top of GrowInk™-T.
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