Sodium Alginate Bioprinting

Sodium alginate is a naturally derived biomaterial that can be chemically crosslinked with calcium chloride to create cell-laden hydrogels. These reagents are used in a variety of applications including bioprinting. The recommended preparation provided in the user instruction below yields a streamlined matrix bioink that supports 3D printed cell-laden constructs. As sodium alginate cannot support itself before it is crosslinked, we recommend using Pluronic F127 or a gelatin bath as a sacrificial support. However, users can modify the preparation to suit their needs. Read on to learn the methods of sodium alginate bioprinting.

Storage and Handling

• Dry Sodium Alginate powder should be stored at room temperature.
• Solubilized Sodium Alginate should be stored at 4ºC in an airtight container.
• Calcium Chloride should be stored at room temperature in a dry environment.

You Will Need

• Sodium alginate
• Calcium chloride
• LifeSupport for FRESH
• Needles
  • We suggest these 0.25″ 30 gauge needles for 2% alginate solutions
  • We suggest these tapered 25 gauge needles for 5% alginate solutions
• Petri dish or well plate for printing
  • Optional: transwell plates can help avoid sample loss or damage throughout experiment. Keep in mind though that transwell membranes may inhibit imaging processes.
• Sterile filters
  • We suggest these for Sodium alginate and Pluronic F127 solutions
  • We suggest these for calcium chloride solutions
• Syringes
  • For the Allevi 1/3/6: 5mL syringes
  • For the Allevi 2: Sterile 10 mL syringes
• Media without calcium or magnesium
  • Sodium alginate can be dissolved in deionized water.
• Deioinized Water
• Sterilized Phosphate Buffered Saline (PBS)

Instructions for Use

Prepare the Sodium Alginate

1. Dissolve 2% or 5% (w/v) sodium alginate in media without calcium or magnesium under stirring at 60°C. Regularly check and mix with spatula to speed up process. (Sodium alginate may be dissolved up to 24-48 hours in advance of use.)
2. *Sterile filter sodium alginate with a 0.45 µm filter into a sterile container.
3. Allow solution to reach 37°C. Mix in desired cell type and concentration. Be sure to pipette vigorously to achieve a homogenous solution.

Prepare Calcium Chloride solution

1. Dissolve 100 mM (0.011 g/ml) concentration of calcium chloride in deionized water. (Calcium chloride solution may be made 24-48 hours in advance of use.)
2. You will need enough calcium chloride to be able to submerge your samples.
3. *Sterile filter calcium chloride solution with a 0.22 µm filter into a sterile container.

Prepare FRESH

For use with alginate, you will suspend FRESH in 0.1 wt% CaCl₂ solution as noted in our protocol on how to prepare FRESH.

Print Settings

	Layer Height (mm)	Speed (mm/s)	Pressure (PSI)	Temperature (°C)	Needle	Crosslinking
Alginate (2%)	0.15	6	7.5	RT	0.25″ straight 30G	Chemical (CaCl2)
Alginate (5%)	0.15	6	15	RT	0.25″ straight 30G	Chemical (CaCl2)

Post-print settings

1. While printing, heat the sterilized solution of CaCl2 to 40-50°C.
2. After the printing has finished, crosslink the finished structures in the heated solution. Crosslink for 15 minutes at room temperature. This will melt the FRESH while chemically crosslinking the alginate.
3. After crosslinking samples, wash each structure 3 times with PBS, then add media.
4. Incubate as needed.

* If not using cells, sterile filtering steps may be skipped.

Notes

1. Adjust gelation time and gel stiffness by varying the concentration of alginate and calcium chloride as well as the crosslinking time. For help adjusting print parameters please contact [email protected].
2. A fill volume change of more than 2 mL may affect pressure settings.

Further Information

• Bioprinted alginate viability results
• Sodium Alginate SDS
• Calcium Chloride SDS
• Sodium alginate troubleshooting guide
• FRESH troubleshooting guide

For further questions about sodium alginate bioprinting or information about these products, please contact [email protected].