8. Repeat step 5 every two layers until completion of the printing
sequence.
9. Once the 3D printing is completed, leave the printed structure
in the secondary crosslinking bath for 10 min.
10. Gently remove the secondary crosslinking solution with a
syringe and replace it with the tertiary crosslinking solution.
11. After 2 min, gently remove the printed structure from the
tertiary crosslinking bath using tweezers.
3.4 Bioprinting of
Cell-laden Alginate
Hydrogel
1. Clean the 3D bioprinter using 70% ethanol and place it inside a
cell culture hood.
2. Heat up the sodium alginate solution and both the primary and
secondary crosslinking solutions to 37
C in a heated bath.
3. Inside of a cell culture hood, load 2 mL of sterile sodium
alginate solution into the extrusion syringe.
4. Load 1 mL of sterile primary crosslinking solution (160 mM
CaCl 2 ) into the extrusion syringe.
5. Load 1 mL of cell suspension into the extrusion syringe.
6. Gently mix the three solutions using a pipet tip until the gel is
formed (2–3 min).
7. Load the syringe in the bioprinter, and continue the printing
process as described in Subheading 3.3, ensuring that you work
in sterility.
8. After printing, transfer the structure to a Petri dish containing
cell culture medium, and place in an incubator at 37
C and 5%
CO 2 . Refresh medium every 2 days.
3.5 Live and Dead
Cell Assay
After printing the cells, it is important to keep an eye out for cell
survival. The following protocol was used for routine cell viability
checks (see Note 8).
1. In a cell culture hood, aspirate the medium from the construct
and replace with PBS.
2. Add 1 μL of propidium iodide stock solution for every mL of
PBS and incubate in the dark at 37
C for 30 min.
3. After incubation, add 0.5 μL fluorescein diacetate stock solution per mL of PBS, and incubate at room temperature for
3–5 min.
4. Aspirate the staining solution and wash the structure once
with PBS.
5. Image the constructs using a fluorescent microscope. Live cells
will be green fluorescent (FITC filter set), dead cells will have
red fluorescent nuclei (TRITC filter set) (see Note 7).
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Atabak Ghanizadeh Tabriz et al.
sequence.
9. Once the 3D printing is completed, leave the printed structure
in the secondary crosslinking bath for 10 min.
10. Gently remove the secondary crosslinking solution with a
syringe and replace it with the tertiary crosslinking solution.
11. After 2 min, gently remove the printed structure from the
tertiary crosslinking bath using tweezers.
3.4 Bioprinting of
Cell-laden Alginate
Hydrogel
1. Clean the 3D bioprinter using 70% ethanol and place it inside a
cell culture hood.
2. Heat up the sodium alginate solution and both the primary and
secondary crosslinking solutions to 37
C in a heated bath.
3. Inside of a cell culture hood, load 2 mL of sterile sodium
alginate solution into the extrusion syringe.
4. Load 1 mL of sterile primary crosslinking solution (160 mM
CaCl 2 ) into the extrusion syringe.
5. Load 1 mL of cell suspension into the extrusion syringe.
6. Gently mix the three solutions using a pipet tip until the gel is
formed (2–3 min).
7. Load the syringe in the bioprinter, and continue the printing
process as described in Subheading 3.3, ensuring that you work
in sterility.
8. After printing, transfer the structure to a Petri dish containing
cell culture medium, and place in an incubator at 37
C and 5%
CO 2 . Refresh medium every 2 days.
3.5 Live and Dead
Cell Assay
After printing the cells, it is important to keep an eye out for cell
survival. The following protocol was used for routine cell viability
checks (see Note 8).
1. In a cell culture hood, aspirate the medium from the construct
and replace with PBS.
2. Add 1 μL of propidium iodide stock solution for every mL of
PBS and incubate in the dark at 37
C for 30 min.
3. After incubation, add 0.5 μL fluorescein diacetate stock solution per mL of PBS, and incubate at room temperature for
3–5 min.
4. Aspirate the staining solution and wash the structure once
with PBS.
5. Image the constructs using a fluorescent microscope. Live cells
will be green fluorescent (FITC filter set), dead cells will have
red fluorescent nuclei (TRITC filter set) (see Note 7).
146
Atabak Ghanizadeh Tabriz et al.