when the stirring suspension starts becoming opaque and
resuming when it has cleared out.
4. Leave Laponite to fully disperse in suspension by continuous
stirring for at least 3 h (Fig. 1; see Note 2). Alginate powder
(600 mg) can then be added to the stirring suspension by slow
addition. Alginate dispersion will be completed by stirring the
suspension for 2 h (see Note 3).
5. Lastly, methylcellulose will be added. Carefully remove the
stirring bar with a sterile spatula, avoiding collecting the suspension and removing excessive Laponite-alginate hydrogel
from the stirrer bar. Insert 600 mg of methylcellulose in the
static Laponite-alginate suspension. Use a sterile spatula
(Fig. 2a) to stir and disperse the methylcellulose powder close
to the top of the suspension (Fig. 2b). Once the methylcellulose powder is dispersed (Fig. 2c), store the 3-3-3 suspension at
4
C overnight (see Note 4).
3.2 Preparation
of Stem Cells
for Printing
1. Skeletal stem cells can be expanded in T175 cm
2 flasks to reach
desired number for studies to be undertaken (see Note 5).
2. Allow cell culture media, PBS, collagenase solution, and trypsin/EDTA to warm up at least 30 min.
3. Remove cell culture media from flasks, wash with PBS, and
insert 10 mL of collagenase working solution in one T175 cm
2
flask. Incubate for 45 min at 37
C, 5% CO 2 (see Note 6).
4. Remove collagenase media, add trypsin/EDTA, and incubate
for 5 min at 37
C, 5% CO 2 . Tap the flasks to allow full stem cell
detachment (see Note 7). Rinse flask with complete α-MEM, to
deactivate trypsin, and collect suspension in a falcon tube.
Centrifuge at 228 RCF for 4 min. Discard supernatant and
resuspend pellet to count stem cells.
5. Cells can be fluorescently labelled using a live-compatible stain,
such as Vybrant DiD (Thermo Fisher Scientific). Prepare a
staining solution at a concentration of 5 μL/mL in FBS-free
medium, and resuspend cells at 1 Â 10
6 cells/mL. Incubate
(37
C, 5% CO 2 ) for 20 min, and then wash three times with
fresh complete cell culture media.
6. Allow 1–5 Â 10
6 cells per gram of clay-based biopaste (see Note
8). Collect the required amount of cell suspension, centrifuge,
and pellet cells.
3.3 Cell-Laden
Clay-Based Bioinks
Printing
1. In a sterile cell culture hood, load 3-3-3 in a pre-weighed falcon
tube. Weigh the paste to obtain 5 g of material. Spin down the
3-3-3 at 755 RCF for 1 min (see Note 9).
2. Resuspend cell pellet in 100 μL complete α-MEM media,
collect cells, and pipette the suspension on top of the 3-3-3.
68
Gianluca Cidonio et al.
resuming when it has cleared out.
4. Leave Laponite to fully disperse in suspension by continuous
stirring for at least 3 h (Fig. 1; see Note 2). Alginate powder
(600 mg) can then be added to the stirring suspension by slow
addition. Alginate dispersion will be completed by stirring the
suspension for 2 h (see Note 3).
5. Lastly, methylcellulose will be added. Carefully remove the
stirring bar with a sterile spatula, avoiding collecting the suspension and removing excessive Laponite-alginate hydrogel
from the stirrer bar. Insert 600 mg of methylcellulose in the
static Laponite-alginate suspension. Use a sterile spatula
(Fig. 2a) to stir and disperse the methylcellulose powder close
to the top of the suspension (Fig. 2b). Once the methylcellulose powder is dispersed (Fig. 2c), store the 3-3-3 suspension at
4
C overnight (see Note 4).
3.2 Preparation
of Stem Cells
for Printing
1. Skeletal stem cells can be expanded in T175 cm
2 flasks to reach
desired number for studies to be undertaken (see Note 5).
2. Allow cell culture media, PBS, collagenase solution, and trypsin/EDTA to warm up at least 30 min.
3. Remove cell culture media from flasks, wash with PBS, and
insert 10 mL of collagenase working solution in one T175 cm
2
flask. Incubate for 45 min at 37
C, 5% CO 2 (see Note 6).
4. Remove collagenase media, add trypsin/EDTA, and incubate
for 5 min at 37
C, 5% CO 2 . Tap the flasks to allow full stem cell
detachment (see Note 7). Rinse flask with complete α-MEM, to
deactivate trypsin, and collect suspension in a falcon tube.
Centrifuge at 228 RCF for 4 min. Discard supernatant and
resuspend pellet to count stem cells.
5. Cells can be fluorescently labelled using a live-compatible stain,
such as Vybrant DiD (Thermo Fisher Scientific). Prepare a
staining solution at a concentration of 5 μL/mL in FBS-free
medium, and resuspend cells at 1 Â 10
6 cells/mL. Incubate
(37
C, 5% CO 2 ) for 20 min, and then wash three times with
fresh complete cell culture media.
6. Allow 1–5 Â 10
6 cells per gram of clay-based biopaste (see Note
8). Collect the required amount of cell suspension, centrifuge,
and pellet cells.
3.3 Cell-Laden
Clay-Based Bioinks
Printing
1. In a sterile cell culture hood, load 3-3-3 in a pre-weighed falcon
tube. Weigh the paste to obtain 5 g of material. Spin down the
3-3-3 at 755 RCF for 1 min (see Note 9).
2. Resuspend cell pellet in 100 μL complete α-MEM media,
collect cells, and pipette the suspension on top of the 3-3-3.
68
Gianluca Cidonio et al.