2.3 Gelatin
Methacrylate (GelMA)
Synthesis
1. Porcine skin gelatin.
2. Dulbecco’s phosphate-buffered saline (DPBS).
3. Methacrylate anhydride (MA).
4. DI water.
5. Dialysis tubing (MWCO 13.5 kDa).
2.4 Photoinitiator
Lithium Phenyl-2,4,6
Trimethylbenzoylphosphinate
(LAP) Synthesis
1. 2,4,6-trimethylbenzoyl chloride.
2. Dimethyl phenylphosphonite.
3. Lithium bromide.
4. 2-butanone.
2.5 Cell and Tissue
Cultures
1. Human umbilical vein endothelial cells (HUVECs).
2. Endothelial cell growth medium (EGM-2, Lonza).
3. C3H/10T1/2 cells (10T1/2 s).
4. Dulbecco’s modified eagle medium (DMEM).
5. Fetal bovine serum (FBS).
6. 0.05% trypsin-EDTA
7. 0.25% trypsin-EDTA
8. DPBS.
2.6 μCOB Setup
1. Digital micromirror array device (DMD, DLP9500UV, Texas
Instrument).
2. UV LED light source (365 nm, Hamamatsu).
3. 3-axis linear stage with motion controller (Newport).
4. Projection optics.
5. Motorized syringe pump.
6. Computer.
2.7 Cell
Viability Assay
1. LIVE/DEAD
® Viability/Cytotoxicity Kit (Invitrogen).
2. DPBS.
2.8 Immunofluorescence Staining
1. Fixation buffer: 4% Paraformaldehyde (PFA) in DPBS.
2. Blocking/permeabilization buffer: 2% bovine serum albumin
(BSA), 0.1% Triton X-100 in DPBS.
3. Anti-CD31 primary antibody: goat anti-human PECAM-1
(M-20, sc-1506, Santa Cruz Biotechnology); 1:100 in blocking/permeabilization buffer. Prepare shortly before use.
4. Anti-alpha-smooth muscle actin primary antibody: rabbit antimouse α-SMA (ab5694, Abcam); 1:100 in blocking/permeabilization buffer. Prepare shortly before use.
5. CF488A donkey anti-rabbit IgG (H+L) (20015, Biotium).
6. CF568 donkey anti-goat IgG (H+L) (20106, Biotium).
Bioprinting of Complex Vascularized Tissues
165
Methacrylate (GelMA)
Synthesis
1. Porcine skin gelatin.
2. Dulbecco’s phosphate-buffered saline (DPBS).
3. Methacrylate anhydride (MA).
4. DI water.
5. Dialysis tubing (MWCO 13.5 kDa).
2.4 Photoinitiator
Lithium Phenyl-2,4,6
Trimethylbenzoylphosphinate
(LAP) Synthesis
1. 2,4,6-trimethylbenzoyl chloride.
2. Dimethyl phenylphosphonite.
3. Lithium bromide.
4. 2-butanone.
2.5 Cell and Tissue
Cultures
1. Human umbilical vein endothelial cells (HUVECs).
2. Endothelial cell growth medium (EGM-2, Lonza).
3. C3H/10T1/2 cells (10T1/2 s).
4. Dulbecco’s modified eagle medium (DMEM).
5. Fetal bovine serum (FBS).
6. 0.05% trypsin-EDTA
7. 0.25% trypsin-EDTA
8. DPBS.
2.6 μCOB Setup
1. Digital micromirror array device (DMD, DLP9500UV, Texas
Instrument).
2. UV LED light source (365 nm, Hamamatsu).
3. 3-axis linear stage with motion controller (Newport).
4. Projection optics.
5. Motorized syringe pump.
6. Computer.
2.7 Cell
Viability Assay
1. LIVE/DEAD
® Viability/Cytotoxicity Kit (Invitrogen).
2. DPBS.
2.8 Immunofluorescence Staining
1. Fixation buffer: 4% Paraformaldehyde (PFA) in DPBS.
2. Blocking/permeabilization buffer: 2% bovine serum albumin
(BSA), 0.1% Triton X-100 in DPBS.
3. Anti-CD31 primary antibody: goat anti-human PECAM-1
(M-20, sc-1506, Santa Cruz Biotechnology); 1:100 in blocking/permeabilization buffer. Prepare shortly before use.
4. Anti-alpha-smooth muscle actin primary antibody: rabbit antimouse α-SMA (ab5694, Abcam); 1:100 in blocking/permeabilization buffer. Prepare shortly before use.
5. CF488A donkey anti-rabbit IgG (H+L) (20015, Biotium).
6. CF568 donkey anti-goat IgG (H+L) (20106, Biotium).
Bioprinting of Complex Vascularized Tissues
165