4. PEG-diacrylate solution: 250–300 mg/mL PEG-DA
(4–20 kDa; see Note 2) in PBS/urea buffer. Freshly prepared.
5. Tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl).
6. Acetone.
7. Dialysis tubing (MWCO ¼ 12–14 kDa).
8. D-Glucose.
2.4 Preparation
of the Bioink
1. Heated magnetic stirrer.
2. Screw top glass vial, preferably of brown glass (see Note 3).
3. Low molecular weight alginate (FMC Biopolymers).
4. GelMA, HAMA, CS-AEMA, or PEG-fibrinogen (see Note 4).
5. 2-Hydroxy-4
0 -(2-hydroxyethoxy)-2-methylpropiophenone
photoinitiator (Irgacure 2959, BASF).
6. HEPES/FBS buffer: 25 mM HEPES buffer containing 10%
v/v fetal bovine serum (FBS).
7. Sterile tubes (1.5–2 mL).
8. 0.2 μm syringe filter.
9. 1 mL syringe with 19G needle.
10. Living cells (according to the application).
11. Cell culture plasticware, media, and consumables.
2.5 Bioprinting
Experiment
1. 3D bioprinter (or three-axis controlled motorized stage).
2. Coaxial needle (see Note 5).
3. Two syringe pumps (see Note 6).
4. UV light source at 365 nm.
5. ~700 μL of sterile bioink (see Subheading 2.4).
6. Crosslinking solution: sterile 300 mM aq. calcium chloride
solution.
7. Two gas-tight glass syringes (1 mL), sterilized by autoclaving.
8. PTFE tubing to connect the syringes to the coaxial needle,
sterilized by autoclaving.
9. Sterile spatula.
10. Sterile plasticware.
11. Cell culture media.
2.6 Immunofluorescence
Evaluation
of Structural
and Functional
Proteins in 3D
Bioprinted Constructs
1. Fixation buffer: 2% paraformaldehyde in PBS.
2. Permeabilization solution: 0.2% v/v Triton X-100 in PBS.
3. Blocking solution: 0.1% v/v Triton X-100, 1% w/v glycine,
10% normal goat serum in PBS.
4. Washing solution: 0.2% v/v Triton X-100 and 1% w/v bovine
serum albumin in PBS.
48
Marco Costantini et al.
(4–20 kDa; see Note 2) in PBS/urea buffer. Freshly prepared.
5. Tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl).
6. Acetone.
7. Dialysis tubing (MWCO ¼ 12–14 kDa).
8. D-Glucose.
2.4 Preparation
of the Bioink
1. Heated magnetic stirrer.
2. Screw top glass vial, preferably of brown glass (see Note 3).
3. Low molecular weight alginate (FMC Biopolymers).
4. GelMA, HAMA, CS-AEMA, or PEG-fibrinogen (see Note 4).
5. 2-Hydroxy-4
0 -(2-hydroxyethoxy)-2-methylpropiophenone
photoinitiator (Irgacure 2959, BASF).
6. HEPES/FBS buffer: 25 mM HEPES buffer containing 10%
v/v fetal bovine serum (FBS).
7. Sterile tubes (1.5–2 mL).
8. 0.2 μm syringe filter.
9. 1 mL syringe with 19G needle.
10. Living cells (according to the application).
11. Cell culture plasticware, media, and consumables.
2.5 Bioprinting
Experiment
1. 3D bioprinter (or three-axis controlled motorized stage).
2. Coaxial needle (see Note 5).
3. Two syringe pumps (see Note 6).
4. UV light source at 365 nm.
5. ~700 μL of sterile bioink (see Subheading 2.4).
6. Crosslinking solution: sterile 300 mM aq. calcium chloride
solution.
7. Two gas-tight glass syringes (1 mL), sterilized by autoclaving.
8. PTFE tubing to connect the syringes to the coaxial needle,
sterilized by autoclaving.
9. Sterile spatula.
10. Sterile plasticware.
11. Cell culture media.
2.6 Immunofluorescence
Evaluation
of Structural
and Functional
Proteins in 3D
Bioprinted Constructs
1. Fixation buffer: 2% paraformaldehyde in PBS.
2. Permeabilization solution: 0.2% v/v Triton X-100 in PBS.
3. Blocking solution: 0.1% v/v Triton X-100, 1% w/v glycine,
10% normal goat serum in PBS.
4. Washing solution: 0.2% v/v Triton X-100 and 1% w/v bovine
serum albumin in PBS.
48
Marco Costantini et al.