Sodium Alginate and Calcium Chloride
The best method for printing with cell-encapsulated sodium alginate is the FRESH method. This simplified method below will allow you to fabricate thin films and very simple constructs with limited resolution. We highly suggest the FRESH method for any complex bioprinting or any bioprinting that requires high resolution with sodium alginate. The protocol below uses Pluronic F127 instead of the FRESH method.
This protocol was written on an Allevi printer with 2 or more extruders. If you have an Allevi 1, just print the pluronic support first, then the alginate infill.
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 as a sacrificial support. However, preparation can be modified by users to suit their needs.
Storage and Handling
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
In addition to sodium alginate and calcium chloride, you will also need:
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.
Sodium alginate can be dissolved in deionized water.
Sterilized Phosphate Buffered Saline (PBS)
*more permanent support such as PCL can also be used.
Instructions for Use
Part 1: Prepare Reagents
1A. Prepare Sodium Alginate
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.)
*Sterile filter sodium alginate with a 0.45 µm filter into a sterile container.
Allow solution to reach 37 °C. Mix in desired cell type and concentration. Be sure to pipette vigorously to achieve a homogenous solution.
1B. Prepare Pluronic F127
*Cool Pluronic F127 to 4°C, then sterile filter with a 0.45 μm filter into a sterile container. (This step may be completed in advance of use.)
Load Pluronic into syringe while still cool.
1C. Prepare Calcium Chloride
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.)
You will need enough calcium chloride to be able to submerge your samples.
*Sterile filter calcium chloride solution with a 0.22 µm filter into a sterile container.
Part 2: Print
2A. Design Files
Prepare your design file through Repetier Host. To put the infill STL inside of the support STL, load both into Repetier Host and click ‘Center Object’ for each. Then upload the sliced gcode file to the Allevi software.
Check out this sodium alginate pluronic example design gcode for reference.
2B. Print File
**Load the sterile pluronic into the left extruder and the cell-laden sodium alginate syringe into the right extruder.
After printing your designs, crosslink finished structures in sterilized calcium chloride solution.
Crosslink for 10-15 minutes at room temperature.
After crosslinking samples, wash each structure 3 times with PBS, then add media.
* If not using cells, sterile filtering steps may be skipped.
** Syringes may be loaded into opposing extruders. Just be sure to match loaded syringes with designated extruders in your gcode file.
|SLICING PARAMETERS||PRINT PARAMETERS|
|Bioink||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)|
|Pluronic||0.15||6||60||RT||0.25" straight 30G||N/A|