10. Cell culture hood.
11. Incubator.
12. Heated bath.
13. Gamma irradiator.
14. Autoclave.
15. Live/dead assay: 6 mg/mL fluorescein stock solution to stain
live cells and 2.5 mM propidium iodide stock solution to stain
dead cells (see Note 5).
3 Methods
3.1 Alginate
Hydrogel and CrossLinking Reagents
Sterilization for 3D
Printing
1. Sterilize the alginate solution by gamma irradiation with 10 Gy
at a rate of 1 Gy/min.
2. Sterilize the cross-linking reagents by autoclaving them at
121
C for 15 min.
3.2 Design and 3D
Printing of Alginate
Hydrogel Structures
1. Prepare the desired 3D CAD and convert it to STL format for
printing.
2. Open the STL file through the relative 3D printer software (see
Notes 6 and 7).
3. Set printing width to 0.35 mm, the printing height to
0.475 mm, and the printing speed to 6 mm/s.
4. Generate the 3D printing file (XDFL format, for the present
setup).
3.3 3D Printing
Procedure
1. Add 80 mM CaCl 2 solution to the alginate solution in a centrifuge tube at a 1:1 volume ratio and shake for 10 s to form a
partially cross-linked alginate hydrogel.
2. Further vortex the gel at 1500 rpm for 30 s.
3. Load some alginate into the extrusion syringe using a 5 mL
syringe.
4. Dampen the nitrocellulose filter paper with the secondary
crosslinking solution and place it into a sterile Petri dish.
5. Purge some gel to ensure the continuous printing of the gel.
6. Open the 3D printing software and execute printing.
7. Once two layers of the 3D structure have been printed, gently
add some secondary crosslinking solution (100 mM CaCl 2 )
into the Petri dish until the level reaches the uppermost surface
of the printed structure. Make sure the secondary crosslinking
bath does not overflow the printed structure (see Note 9).
Bioprinting of Complex Alginate Constructs
145
11. Incubator.
12. Heated bath.
13. Gamma irradiator.
14. Autoclave.
15. Live/dead assay: 6 mg/mL fluorescein stock solution to stain
live cells and 2.5 mM propidium iodide stock solution to stain
dead cells (see Note 5).
3 Methods
3.1 Alginate
Hydrogel and CrossLinking Reagents
Sterilization for 3D
Printing
1. Sterilize the alginate solution by gamma irradiation with 10 Gy
at a rate of 1 Gy/min.
2. Sterilize the cross-linking reagents by autoclaving them at
121
C for 15 min.
3.2 Design and 3D
Printing of Alginate
Hydrogel Structures
1. Prepare the desired 3D CAD and convert it to STL format for
printing.
2. Open the STL file through the relative 3D printer software (see
Notes 6 and 7).
3. Set printing width to 0.35 mm, the printing height to
0.475 mm, and the printing speed to 6 mm/s.
4. Generate the 3D printing file (XDFL format, for the present
setup).
3.3 3D Printing
Procedure
1. Add 80 mM CaCl 2 solution to the alginate solution in a centrifuge tube at a 1:1 volume ratio and shake for 10 s to form a
partially cross-linked alginate hydrogel.
2. Further vortex the gel at 1500 rpm for 30 s.
3. Load some alginate into the extrusion syringe using a 5 mL
syringe.
4. Dampen the nitrocellulose filter paper with the secondary
crosslinking solution and place it into a sterile Petri dish.
5. Purge some gel to ensure the continuous printing of the gel.
6. Open the 3D printing software and execute printing.
7. Once two layers of the 3D structure have been printed, gently
add some secondary crosslinking solution (100 mM CaCl 2 )
into the Petri dish until the level reaches the uppermost surface
of the printed structure. Make sure the secondary crosslinking
bath does not overflow the printed structure (see Note 9).
Bioprinting of Complex Alginate Constructs
145