Use a sterile spatula to gently mix thoroughly for at least 1 min
(see Note 10).
3. Load the cell-laden clay-based bioink in a Luer-lock syringe.
10 or 20 mL syringes are to be preferred, as they have a wider
aperture to allow easier biopaste loading (see Note 11).
4. Syringe plunger can be inserted, pushing 3-3-3 toward the tip.
Lock in a sterile Luer-lock conical nozzle of correct dimension
for cell printing (see Note 12).
5. Wipe 3D printer depositing surface, syringe holder, and piston
with 70% ethanol for sterile cell printing.
6. Lattice 0/90
can be readily printed (Fig. 3a) with stem cellladen 3-3-3 using a pressure between 80 and 95 kPa, depending on printer setup, pressure-driven adaptors, pressure system,
and printer mechanics (see Note 13).
7. Several structures can be produced by 3-3-3 printing as
detailed in our recent publication [16]. A 12 Â 12 mm construct (Fig. 3b, c) can be readily printed with a single-arm
piston-driven extrusion-based bioprinter (see Note 14).
8. Post-printing, crosslink scaffolds with calcium chloride solution for 10 min. Remove calcium chloride solution, and cultivate cell-laden scaffolds in complete medium for long-term
culture at 37
C, 5% CO 2 , changing medium every 2 days (see
Note 15).
9. If cell viability investigation is needed, this can be performed
with calcein-AM staining. To stain constructs at time points,
use 0.6 μL calcein AM per mL of FBS-free medium. Incubate
(37
C, 5% CO 2 ) for 1 h. Remove medium, and wash with
HBSS at least twice. Image with fluorescence or confocal
microscope (Fig. 3d).
4 Notes
1. It is crucial for clay-based composite hydrogels to be prepared
from DW with specific resistivity (18.2 MΩ cm) showing signs
of coagulation or precipitation at higher and lower resistivity
value, respectively.
2. It is crucial that Laponite suspension is left for 3 h to stir
continuously at room temperature at 14 RCF. This allows
Laponite nanodiscs to disperse homogenously resulting in a
clear solution. If Laponite is not allowed to disperse, it will
aggregate and eventually fail to intercalate with alginate and
methylcellulose polymeric chain.
3. Powder may aggregate at the air-liquid interface. By momentarily raising the stirring speed to 28 RCF, the alginate
Clay Bioinks for Skeletal Regeneration
69
(see Note 10).
3. Load the cell-laden clay-based bioink in a Luer-lock syringe.
10 or 20 mL syringes are to be preferred, as they have a wider
aperture to allow easier biopaste loading (see Note 11).
4. Syringe plunger can be inserted, pushing 3-3-3 toward the tip.
Lock in a sterile Luer-lock conical nozzle of correct dimension
for cell printing (see Note 12).
5. Wipe 3D printer depositing surface, syringe holder, and piston
with 70% ethanol for sterile cell printing.
6. Lattice 0/90
can be readily printed (Fig. 3a) with stem cellladen 3-3-3 using a pressure between 80 and 95 kPa, depending on printer setup, pressure-driven adaptors, pressure system,
and printer mechanics (see Note 13).
7. Several structures can be produced by 3-3-3 printing as
detailed in our recent publication [16]. A 12 Â 12 mm construct (Fig. 3b, c) can be readily printed with a single-arm
piston-driven extrusion-based bioprinter (see Note 14).
8. Post-printing, crosslink scaffolds with calcium chloride solution for 10 min. Remove calcium chloride solution, and cultivate cell-laden scaffolds in complete medium for long-term
culture at 37
C, 5% CO 2 , changing medium every 2 days (see
Note 15).
9. If cell viability investigation is needed, this can be performed
with calcein-AM staining. To stain constructs at time points,
use 0.6 μL calcein AM per mL of FBS-free medium. Incubate
(37
C, 5% CO 2 ) for 1 h. Remove medium, and wash with
HBSS at least twice. Image with fluorescence or confocal
microscope (Fig. 3d).
4 Notes
1. It is crucial for clay-based composite hydrogels to be prepared
from DW with specific resistivity (18.2 MΩ cm) showing signs
of coagulation or precipitation at higher and lower resistivity
value, respectively.
2. It is crucial that Laponite suspension is left for 3 h to stir
continuously at room temperature at 14 RCF. This allows
Laponite nanodiscs to disperse homogenously resulting in a
clear solution. If Laponite is not allowed to disperse, it will
aggregate and eventually fail to intercalate with alginate and
methylcellulose polymeric chain.
3. Powder may aggregate at the air-liquid interface. By momentarily raising the stirring speed to 28 RCF, the alginate
Clay Bioinks for Skeletal Regeneration
69