9. Remove the secondary antibody solution and rinse the tissue
construct with DPBS three times with DPBS for 5 min each.
10. Image the tissue construct with confocal microscopy (Fig. 2).
4 Notes
1. Add the TEA dropwise with a syringe to make sure it is thoroughly mixed.
2. Add the GM dropwise with a syringe to make sure it is thoroughly mixed.
3. Cover the reaction solution from light with aluminum foil.
4. Keep stirring the DI water during dialysis. Change the DI water
after 2 h, 4 h, and 24 h.
5. Freeze the solution at an angle to maximize surface area for
subsequent lyophilization.
6. Use a water bath to control the temperature at 60
C and allow
the gelatin to completely dissolve before proceeding to the
next step.
7. Fill the reaction container with argon during the reaction.
8. Warm the DPBS to 50
C prior to the end of the 3 h.
9. Keep stirring the DI water during dialysis. Change the DI water
2–3 times a day. Heat the fresh DI water to 40
C before the
change.
10. Use passage 3–6 HUVECs for bioprinting.
11. Change the medium every other day and use a 1 mL pipette
instead of the vacuum line to remove the old medium.
12. Incubate the cells in trypsin-EDTA solution for 5 min at 37
C
in the CO 2 incubator.
13. The cell concentrations here are twice the final concentrations.
This cell solution will be mixed with the prepolymer solution at
a volume ratio of 1:1.
14. Dissolve GelMA by heating the solution at 37
C. Do not
vortex the GelMA solution to avoid bubbles.
15. Gently pipette the mixture of the prepolymer and the cells to
make a homogenous solution and avoid creating bubbles.
16. The live dead working solution should be used within 1 day,
since the calcein AM aqueous solution is susceptible to
hydrolysis.
17. Serum esterase present in the serum-supplemented media can
hydrolyze the calcein AM and cause increase in extracellular
fluorescence. Therefore, it is important to wash the tissue
constructs well and gently with 500–1000 volumes of DPBS.
Bioprinting of Complex Vascularized Tissues
171
construct with DPBS three times with DPBS for 5 min each.
10. Image the tissue construct with confocal microscopy (Fig. 2).
4 Notes
1. Add the TEA dropwise with a syringe to make sure it is thoroughly mixed.
2. Add the GM dropwise with a syringe to make sure it is thoroughly mixed.
3. Cover the reaction solution from light with aluminum foil.
4. Keep stirring the DI water during dialysis. Change the DI water
after 2 h, 4 h, and 24 h.
5. Freeze the solution at an angle to maximize surface area for
subsequent lyophilization.
6. Use a water bath to control the temperature at 60
C and allow
the gelatin to completely dissolve before proceeding to the
next step.
7. Fill the reaction container with argon during the reaction.
8. Warm the DPBS to 50
C prior to the end of the 3 h.
9. Keep stirring the DI water during dialysis. Change the DI water
2–3 times a day. Heat the fresh DI water to 40
C before the
change.
10. Use passage 3–6 HUVECs for bioprinting.
11. Change the medium every other day and use a 1 mL pipette
instead of the vacuum line to remove the old medium.
12. Incubate the cells in trypsin-EDTA solution for 5 min at 37
C
in the CO 2 incubator.
13. The cell concentrations here are twice the final concentrations.
This cell solution will be mixed with the prepolymer solution at
a volume ratio of 1:1.
14. Dissolve GelMA by heating the solution at 37
C. Do not
vortex the GelMA solution to avoid bubbles.
15. Gently pipette the mixture of the prepolymer and the cells to
make a homogenous solution and avoid creating bubbles.
16. The live dead working solution should be used within 1 day,
since the calcein AM aqueous solution is susceptible to
hydrolysis.
17. Serum esterase present in the serum-supplemented media can
hydrolyze the calcein AM and cause increase in extracellular
fluorescence. Therefore, it is important to wash the tissue
constructs well and gently with 500–1000 volumes of DPBS.
Bioprinting of Complex Vascularized Tissues
171