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.
Figure 1: Live/dead images of human neonatal dermal fibroblasts encapsulated in 2% alginate (left) and a piece of a bioprinted cell-laden 2% alginate ring after 7 days of culture (right).
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:
- Pluronic F127*
- 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
- Sterile 10 ml BD syringes
- Media without calcium or magnesium
- Sodium alginate can be dissolved in deionized water.
- Deioinized 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 solution.
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. Sterilize 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. 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, then upload the sliced gcode file to biobots software.
Check out this sodium alginate pluronic example design gcode for reference.
2b. **Load the cell-laden sodium alginate syringe into the right extruder and the pluronic F127 syringe into the left extruder.
2c. After printing your designs, crosslink finished structures in sterilized calcium chloride solution.
Crosslink for 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.
|Extruder||Speed (mm/s)||Layer height (mm)||Nozzle Diam (mm)||Gauge|
1 (Left- Pluronic F127)
2 (Right – Sodium Alginate 2%)
2(Right – Sodium Alginate 5%)
|Extruder||Pressure (psi)||Print Temp (°C)||Volume Fill (ml)|
1 (Pluronic F127)
2 (Sodium Alginate 2%)
2 (Sodium Alginate 5%)
These materials do not need to be photocrosslinked, so all of the crosslink settings may be set to 0.
- Gelation time and gel stiffness can be adjusted by varying the concentration of alginate and calcium chloride as well as the crosslinking time. For help adjusting print parameters please contact email@example.com.
- A fill volume change of more than 2 ml may affect pressure settings.
- A lower gauge size or tapered gauge will require lower pressure, while a higher gauge will require higher pressure for extrusion. Lowering the gauge size will also generally lower resolution.
for further questions or information about these products, please contact firstname.lastname@example.org