3. Phosphate buffer saline (Dulbecco’s phosphate-buffered saline
(PBS) without calcium and magnesium).
4. Complete medium: α-MEM (alpha-minimum essential
medium with L-glutamine) containing 10% fetal bovine serum
(FBS) and 1% penicillin-streptomycin mixture (10,000 U/mL
penicillin/mL, 10,000 U/mL streptomycin).
5. Collagenase working solution: prepare a 200 μg/mL solution
of collagenase (collagenase type I from Clostridium histolyticum, sterile-filtered) in serum-free α-MEM.
6. Trypsin/EDTA (trypsin/ethylenediaminetetraacetic acid, for
cell culture).
7. Vybrant DiD (Thermo Fisher Scientific) cell-labelling solution.
2.3 Bioprinting
1. Luer-lock syringe, 10–20 mL volume.
2. Sterile spatula for paste loading.
3. Luer-lock nozzle tips (22 gauge tapered dispensing tip
(ID 0.41 mm) (sterilized in ethanol or by gamma-irradiation).
4. Extrusion-based bioprinter, able to generate 80–95 kPa pressure on biopaste (biopaste has been tested in pneumatic [16]
and piston-driven extrusion printer).
5. Six-well tissue culture plate or, alternatively, 35 mm Petri
dishes.
2.4 Post-printing
Processing
1. Calcium chloride solution: 100 mM calcium chloride (CaCl 2 )
in DW.
2. Sterile spatulas.
3. Sterile 12-well plates containing α-MEM to transfer scaffolds
after crosslinking.
4. Hank’s Balanced Salt Solution (HBSS, calcium, magnesium).
3 Methods
Perform the following procedures at room temperature in a cell
culture hood with laminar flow when synthesizing biopaste for cell
printing.
3.1 Biopaste
Synthesis
1. Separately autoclave proper amounts of biomaterial powders
(Laponite, alginate, and methylcellulose) in glass bottles
with lid.
2. Add 20 mL DW to a sterile beaker with magnetic bar.
3. Keeping the solution under magnetic stirring at 14 RCF,
slowly add 600 mg of sterile nanosilicate Laponite powder.
Perform a slow and gentle addition of the powder, pausing
Clay Bioinks for Skeletal Regeneration
67
(PBS) without calcium and magnesium).
4. Complete medium: α-MEM (alpha-minimum essential
medium with L-glutamine) containing 10% fetal bovine serum
(FBS) and 1% penicillin-streptomycin mixture (10,000 U/mL
penicillin/mL, 10,000 U/mL streptomycin).
5. Collagenase working solution: prepare a 200 μg/mL solution
of collagenase (collagenase type I from Clostridium histolyticum, sterile-filtered) in serum-free α-MEM.
6. Trypsin/EDTA (trypsin/ethylenediaminetetraacetic acid, for
cell culture).
7. Vybrant DiD (Thermo Fisher Scientific) cell-labelling solution.
2.3 Bioprinting
1. Luer-lock syringe, 10–20 mL volume.
2. Sterile spatula for paste loading.
3. Luer-lock nozzle tips (22 gauge tapered dispensing tip
(ID 0.41 mm) (sterilized in ethanol or by gamma-irradiation).
4. Extrusion-based bioprinter, able to generate 80–95 kPa pressure on biopaste (biopaste has been tested in pneumatic [16]
and piston-driven extrusion printer).
5. Six-well tissue culture plate or, alternatively, 35 mm Petri
dishes.
2.4 Post-printing
Processing
1. Calcium chloride solution: 100 mM calcium chloride (CaCl 2 )
in DW.
2. Sterile spatulas.
3. Sterile 12-well plates containing α-MEM to transfer scaffolds
after crosslinking.
4. Hank’s Balanced Salt Solution (HBSS, calcium, magnesium).
3 Methods
Perform the following procedures at room temperature in a cell
culture hood with laminar flow when synthesizing biopaste for cell
printing.
3.1 Biopaste
Synthesis
1. Separately autoclave proper amounts of biomaterial powders
(Laponite, alginate, and methylcellulose) in glass bottles
with lid.
2. Add 20 mL DW to a sterile beaker with magnetic bar.
3. Keeping the solution under magnetic stirring at 14 RCF,
slowly add 600 mg of sterile nanosilicate Laponite powder.
Perform a slow and gentle addition of the powder, pausing
Clay Bioinks for Skeletal Regeneration
67