5. Primary antibodies.
6. Fluorophore-conjugated secondary antibodies.
7. DAPI nuclear stain (1:10000).
8. Widefield or confocal fluorescence microscope.
3 Methods
3.1 Synthesis
of Gelatin
Methacrylate (GelMA)
and Hyaluronic Acid
Methacrylate (HAMA)
3.1.1 Gelatin
Methacrylate
1. Dissolve 1 g of gelatin in 10 mL of PBS under magnetic stirring
at 50
C.
2. Dropwise add 0.8 mL of MA under vigorous stirring. The
reaction mixture must be kept at 50
C.
3. Stop the reaction after 2 h.
4. Dilute the reaction mixture to 100 mL, transfer it into dialysis
tubes, and dialyze at 40
C against distilled water for 3 days
with dialysate changes three times per day.
5. Transfer the solution into round-bottom flasks and freeze-dry.
3.1.2 Hyaluronic
Methacrylate
1. Dissolve 1 g of HA in 100 mL of DIW under magnetic stirring
at room temperature.
2. Dropwise add 20-fold excess of MA vs. the disaccharide repeating unit (2-acetamido-2-deoxy-α-D-glucose and β-D-glucuronic acid; residue MW, 401). For a solution containing 1 g of
HA, this corresponds to a volume of 0.37 mL.
3. During MA addition, continuously adjust the pH of the reaction mixture to 8.0 by adding aliquots of 5 M NaOH solution.
4. Leave the reaction mixture under stirring on ice for 24 h.
5. Perform dialysis at room temperature for 3 days with dialysate
changes three times per day, followed by freeze drying.
3.1.3 Synthesis
of Chondroitin Sulfate
2-Aminoethyl Methacrylate
(CS-AEMA)
1. Dissolve 1 g of chondroitin sulfate under magnetic stirring at
room temperature in 100 mL of MES buffer containing 0.5 M
of NaCl.
3. Sequentially add 0.27 g of NHS and 0.85 g EDC and, after
ca. 5 min, 0.38 g AEMA.
3. Leave the reaction mixture under stirring for 24 h at room
temperature.
4. Follow step 5 of Subheading 3.1.2.
Photocurable Biopolymers for Coaxial Bioprinting
49
6. Fluorophore-conjugated secondary antibodies.
7. DAPI nuclear stain (1:10000).
8. Widefield or confocal fluorescence microscope.
3 Methods
3.1 Synthesis
of Gelatin
Methacrylate (GelMA)
and Hyaluronic Acid
Methacrylate (HAMA)
3.1.1 Gelatin
Methacrylate
1. Dissolve 1 g of gelatin in 10 mL of PBS under magnetic stirring
at 50
C.
2. Dropwise add 0.8 mL of MA under vigorous stirring. The
reaction mixture must be kept at 50
C.
3. Stop the reaction after 2 h.
4. Dilute the reaction mixture to 100 mL, transfer it into dialysis
tubes, and dialyze at 40
C against distilled water for 3 days
with dialysate changes three times per day.
5. Transfer the solution into round-bottom flasks and freeze-dry.
3.1.2 Hyaluronic
Methacrylate
1. Dissolve 1 g of HA in 100 mL of DIW under magnetic stirring
at room temperature.
2. Dropwise add 20-fold excess of MA vs. the disaccharide repeating unit (2-acetamido-2-deoxy-α-D-glucose and β-D-glucuronic acid; residue MW, 401). For a solution containing 1 g of
HA, this corresponds to a volume of 0.37 mL.
3. During MA addition, continuously adjust the pH of the reaction mixture to 8.0 by adding aliquots of 5 M NaOH solution.
4. Leave the reaction mixture under stirring on ice for 24 h.
5. Perform dialysis at room temperature for 3 days with dialysate
changes three times per day, followed by freeze drying.
3.1.3 Synthesis
of Chondroitin Sulfate
2-Aminoethyl Methacrylate
(CS-AEMA)
1. Dissolve 1 g of chondroitin sulfate under magnetic stirring at
room temperature in 100 mL of MES buffer containing 0.5 M
of NaCl.
3. Sequentially add 0.27 g of NHS and 0.85 g EDC and, after
ca. 5 min, 0.38 g AEMA.
3. Leave the reaction mixture under stirring for 24 h at room
temperature.
4. Follow step 5 of Subheading 3.1.2.
Photocurable Biopolymers for Coaxial Bioprinting
49