3.1.4 Synthesis of
PEG(monoacrylate)Fibrinogen
1. Prepare 100 mL of a 7 mg/mL solution of fibrinogen in
PBS/urea buffer.
2. Add TCEP-HCl at a molar ratio 68:1 with respect to fibrinogen cysteines, and leave the solution under stirring for 15 min
at 25
C until complete dissolution.
3. Add the PEG-DA solution so that the PEG-fibrinogen molar
ratio is 145:1.
4. Leave the reaction overnight in the dark at 25
C.
5. Precipitate the final PEGylated fibrinogen product in fivefold
excess acetone at room temperature for 20 min under stirring.
6. Centrifuge at 4000 Â g for 20 min.
7. Redissolve the resulting pellet to 20 mg/mL protein concentration in PBS/urea buffer.
8. Dialyze the PEGylated fibrinogen against PBS at 4
C for
2 days with twice-daily changes.
9. Lyophilize and store the PEGylated product in argon at
À80
C.
3.2 Preparation
of the Bioink and Cell
Resuspension
3.2.1 Preparation
of the Bioink
1. Weigh 40 mg of alginate (see Note 7) in a 2 mL tube.
2. Weigh the photocurable biopolymer(s) to be added to the
bioink (see Note 8).
3. Add 1 mL of HEPES/FBS buffer (see Note 9).
4. Vortex until dissolution.
5. Centrifuge to remove air bubbles.
6. Filter the bioink using 0.22 μm syringe filters in a laminar
flow hood.
3.2.2 Resuspension
of the Cells Within
the Bioink
The following instructions must be conducted in a laminar flow
hood.
1. Following standard procedures, detach adherent cells, resuspend them in suitable culture medium, and calculate cell
concentration.
2. Place the volume of cell suspension containing the desired
number of cells in a sterile 2 mL tube, and centrifuge for
5 min at 180 Â g (see Note 10).
3. Gently remove the supernatant (see Note 11).
4. Add 1 mL of sterile-filtered bioink solution and gently resuspend the cells.
3.3 Bioprinting
Experiment
Bioprinting should be performed within a biological safety hood.
1. Create a G-code for the desired construct geometry (see Note
12).
50
Marco Costantini et al.
PEG(monoacrylate)Fibrinogen
1. Prepare 100 mL of a 7 mg/mL solution of fibrinogen in
PBS/urea buffer.
2. Add TCEP-HCl at a molar ratio 68:1 with respect to fibrinogen cysteines, and leave the solution under stirring for 15 min
at 25
C until complete dissolution.
3. Add the PEG-DA solution so that the PEG-fibrinogen molar
ratio is 145:1.
4. Leave the reaction overnight in the dark at 25
C.
5. Precipitate the final PEGylated fibrinogen product in fivefold
excess acetone at room temperature for 20 min under stirring.
6. Centrifuge at 4000 Â g for 20 min.
7. Redissolve the resulting pellet to 20 mg/mL protein concentration in PBS/urea buffer.
8. Dialyze the PEGylated fibrinogen against PBS at 4
C for
2 days with twice-daily changes.
9. Lyophilize and store the PEGylated product in argon at
À80
C.
3.2 Preparation
of the Bioink and Cell
Resuspension
3.2.1 Preparation
of the Bioink
1. Weigh 40 mg of alginate (see Note 7) in a 2 mL tube.
2. Weigh the photocurable biopolymer(s) to be added to the
bioink (see Note 8).
3. Add 1 mL of HEPES/FBS buffer (see Note 9).
4. Vortex until dissolution.
5. Centrifuge to remove air bubbles.
6. Filter the bioink using 0.22 μm syringe filters in a laminar
flow hood.
3.2.2 Resuspension
of the Cells Within
the Bioink
The following instructions must be conducted in a laminar flow
hood.
1. Following standard procedures, detach adherent cells, resuspend them in suitable culture medium, and calculate cell
concentration.
2. Place the volume of cell suspension containing the desired
number of cells in a sterile 2 mL tube, and centrifuge for
5 min at 180 Â g (see Note 10).
3. Gently remove the supernatant (see Note 11).
4. Add 1 mL of sterile-filtered bioink solution and gently resuspend the cells.
3.3 Bioprinting
Experiment
Bioprinting should be performed within a biological safety hood.
1. Create a G-code for the desired construct geometry (see Note
12).
50
Marco Costantini et al.