9. Mix prepolymer B and cell solution at a volume ratio of 1:1 to
get a final composition of 2.5% (w/v) GelMA, 1%(w/v)
GM-HA, 0.15%(w/v) LAP, 20 million HUVECs/mL, and
40,000 10T1/2 s/mL (see Note 15).
10. Load the cell-prepolymer mixture on top of the base layer and
the second mask in the middle in Fig. 1 onto the DMD chip.
11. Expose the UV light for 15 s to print the vascular layer.
Remove the unpolymerized part of the cell-prepolymer mixture and wash with DPBS.
12. Load prepolymer A onto the fabrication stage and the third
mask on the right in Fig. 1 onto the DMD chip.
13. Expose the UV light for 15 s to print the top layer to enclose
the vascular network. Remove the non-polymerized part of the
cell-prepolymer mixture and wash with DPBS.
14. Transfer the printed vascularized tissue construct to a well plate
and culture it with EGM-2.
3.7 Cell Viability Test
1. Cell viability is performed on day 1, day 3, and day 7 after
printing.
2. Prepare the live/dead assay by mixing a final concentration of
2 μM calcein AM (live cell stain) and 4 μM ethidium
homodimer-1 (dead cell stain) solution with DPBS and vortex
to mix thoroughly (see Note 16).
3. Remove the culture medium and wash the tissue construct with
DPBS three times (see Note 17).
4. Add the live/dead working solution to the tissue construct and
incubate at room temperature for 30 min (see Note 18).
5. Remove the live/dead assay after the incubation and add DPBS
to the tissue construct.
6. Take fluorescence images of the tissue construct at different
heights (Z positions) (see Note 19).
7. Cell viability can be calculated as the ratio between live cells and
total cells (see Note 20).
3.8 Immunofluorescence Staining
1. Remove the culture media and fix the tissue construct immediately with fixation buffer for 30 min at room temperature.
2. Remove the fixation buffer and rinse the tissue construct with
DPBS three times for 5 min each.
3. Treat the samples with blocking and permeabilization buffer
for 1 h at room temperature.
4. Near the end of the blocking and permeabilization process,
prepare primary antibody solution by diluting CD31 and
α-SMA in the 2% BSA and 0.1% Triton X-100 solution both
at a ratio of 1:100.
Bioprinting of Complex Vascularized Tissues
169
get a final composition of 2.5% (w/v) GelMA, 1%(w/v)
GM-HA, 0.15%(w/v) LAP, 20 million HUVECs/mL, and
40,000 10T1/2 s/mL (see Note 15).
10. Load the cell-prepolymer mixture on top of the base layer and
the second mask in the middle in Fig. 1 onto the DMD chip.
11. Expose the UV light for 15 s to print the vascular layer.
Remove the unpolymerized part of the cell-prepolymer mixture and wash with DPBS.
12. Load prepolymer A onto the fabrication stage and the third
mask on the right in Fig. 1 onto the DMD chip.
13. Expose the UV light for 15 s to print the top layer to enclose
the vascular network. Remove the non-polymerized part of the
cell-prepolymer mixture and wash with DPBS.
14. Transfer the printed vascularized tissue construct to a well plate
and culture it with EGM-2.
3.7 Cell Viability Test
1. Cell viability is performed on day 1, day 3, and day 7 after
printing.
2. Prepare the live/dead assay by mixing a final concentration of
2 μM calcein AM (live cell stain) and 4 μM ethidium
homodimer-1 (dead cell stain) solution with DPBS and vortex
to mix thoroughly (see Note 16).
3. Remove the culture medium and wash the tissue construct with
DPBS three times (see Note 17).
4. Add the live/dead working solution to the tissue construct and
incubate at room temperature for 30 min (see Note 18).
5. Remove the live/dead assay after the incubation and add DPBS
to the tissue construct.
6. Take fluorescence images of the tissue construct at different
heights (Z positions) (see Note 19).
7. Cell viability can be calculated as the ratio between live cells and
total cells (see Note 20).
3.8 Immunofluorescence Staining
1. Remove the culture media and fix the tissue construct immediately with fixation buffer for 30 min at room temperature.
2. Remove the fixation buffer and rinse the tissue construct with
DPBS three times for 5 min each.
3. Treat the samples with blocking and permeabilization buffer
for 1 h at room temperature.
4. Near the end of the blocking and permeabilization process,
prepare primary antibody solution by diluting CD31 and
α-SMA in the 2% BSA and 0.1% Triton X-100 solution both
at a ratio of 1:100.
Bioprinting of Complex Vascularized Tissues
169