2. Connect the coaxial needle to the Z-axis of the bioprinter/
three-axis coordinated motion control system.
3. Calibrate the Z-axis (i.e., set the zero level of the axis on top of
the surface you will use to print).
4. Fill a 1 mL syringe with the bioink.
5. Fill another 1 mL syringe with the crosslinking solution.
6. Mount the two syringes on the syringe pumps and connect
them to the inner/outer needles of the coaxial assembly using
PTFE tubing.
7. Start the flow of the two pumps.
8. Start the G-code.
9. After bioprinting, collect your deposited sample with a sterile
spatula and place in a Petri dish.
10. Perform photo-crosslinking under UV light (see Note 13).
11. After UV crosslinking, take your sample and place in a multiwell plate and add the desired cell culture medium.
3.4 Immunofluorescence
Evaluation
of Structural
and Functional
Proteins in 3D
Bioprinted Constructs
The following protocol for immunofluorescence staining has been
established for the processing of bioprinted constructs and can be
further optimized according to the specific application.
1. Wash constructs in PBS 5 min at RT.
2. Fix constructs in fixation buffer for 10 min at 4
C (see Note
14).
3. Wash constructs in PBS for PFA excess removal 5 min at RT.
4. Permeabilize in permeabilizing solution for 1 h at RT.
5. Incubate in blocking solution for 1 h at RT.
6. Incubate with primary antibodies (dilution according to the
manufacturer’s instructions) in blocking solution (1 h at RT or
o/n at 4
C).
7. Wash constructs in washing solution twice for 15 and 5 min
at RT.
8. Incubate with secondary antibodies (dilution according to the
manufacturer’s instructions) in blocking solution for 1 h at RT.
9. Wash constructs twice in washing solution for 10 min and once
in PBS for 10 min at RT.
10. Incubate with DAPI (1:10000) for 10 min at RT to label
nuclei.
11. Mount constructs with Aqua-Poly/Mount (Polysciences)
mounting medium.
12. Specimens can be observed under a widefield or confocal fluorescence microscope.
Photocurable Biopolymers for Coaxial Bioprinting
51
three-axis coordinated motion control system.
3. Calibrate the Z-axis (i.e., set the zero level of the axis on top of
the surface you will use to print).
4. Fill a 1 mL syringe with the bioink.
5. Fill another 1 mL syringe with the crosslinking solution.
6. Mount the two syringes on the syringe pumps and connect
them to the inner/outer needles of the coaxial assembly using
PTFE tubing.
7. Start the flow of the two pumps.
8. Start the G-code.
9. After bioprinting, collect your deposited sample with a sterile
spatula and place in a Petri dish.
10. Perform photo-crosslinking under UV light (see Note 13).
11. After UV crosslinking, take your sample and place in a multiwell plate and add the desired cell culture medium.
3.4 Immunofluorescence
Evaluation
of Structural
and Functional
Proteins in 3D
Bioprinted Constructs
The following protocol for immunofluorescence staining has been
established for the processing of bioprinted constructs and can be
further optimized according to the specific application.
1. Wash constructs in PBS 5 min at RT.
2. Fix constructs in fixation buffer for 10 min at 4
C (see Note
14).
3. Wash constructs in PBS for PFA excess removal 5 min at RT.
4. Permeabilize in permeabilizing solution for 1 h at RT.
5. Incubate in blocking solution for 1 h at RT.
6. Incubate with primary antibodies (dilution according to the
manufacturer’s instructions) in blocking solution (1 h at RT or
o/n at 4
C).
7. Wash constructs in washing solution twice for 15 and 5 min
at RT.
8. Incubate with secondary antibodies (dilution according to the
manufacturer’s instructions) in blocking solution for 1 h at RT.
9. Wash constructs twice in washing solution for 10 min and once
in PBS for 10 min at RT.
10. Incubate with DAPI (1:10000) for 10 min at RT to label
nuclei.
11. Mount constructs with Aqua-Poly/Mount (Polysciences)
mounting medium.
12. Specimens can be observed under a widefield or confocal fluorescence microscope.
Photocurable Biopolymers for Coaxial Bioprinting
51