Granular hydrogels are an exciting new class of biomaterials with many advantageous properties. Our hope is that in sharing these methods, we can increase the access and innovation in granular hydrogels for biomedical applications. This technique is simple, low-cost, easily adaptable to many chemistries and can be implemented in virtually any laboratory.
Load a 3-milliliter syringe with the hydrogel precursor solution. Remove the plunger from the back of an empty 3-milliliter syringe and add a tip cap to the top of the syringe barrel. Use a 1, 000-microliters pipette to transfer the hydrogel precursor solution into the syringe barrel with the tip cap.
Hold the syringe barrel with hydrogel precursor solution in one hand with the tip cap facing down and the open end of the barrel facing up. Return the syringe plunger to the opening of the back of the syringe barrel. Gently push the syringe plunger into the barrel just enough to seal the opening at the back of the syringe barrel, carefully holding the plunger and syringe barrel together to ensure the back of the syringe barrel is sealed with the plunger.
Invert the syringe such that the plunger is facing down and the tip cap is now facing up. Remove the tip cap and gently push the plunger into the syringe barrel until all the air is removed from the syringe. Reattach the tip cap to the syringe.
Ensure that the hydrogel precursor solution is secured within the 3-milliliter syringe with a tip cap. Ensure proper personal protective equipment and safeguards are taken prior to turning on the UV lamp. Place the 3-milliliter syringe loaded with the hydrogel precursor solution under the UV spot cure lamp for a desired amount of time to fully photo-crosslink.
Turn off the UV lamp and remove the syringe. Ensure that the hydrogel is now photo-crosslinked within the syringe. Remove the plunger from the back of an empty 3-milliliter syringe.
Secure a tip cap to the luer-lock. Remove the tip cap from the syringe containing the photo-crosslinked bulk hydrogel. Line up the top of the hydrogel syringe with the opening of the barrel on the empty syringe.
Extrude the bulk hydrogel through the syringe opening into the barrel of the empty syringe. Hold the syringe that contains the extruded hydrogel such that the tip cap is facing down and the barrel opening is facing up. Using a 1, 000-microliters pipette, add 1.5 microliters of PBS to the syringe barrel.
Align the syringe plunger with the opening of the barrel, just barely pushing the plunger in enough to create a seal. Invert the syringe such that the plunger is now facing down and the tip cap is facing up, making sure to hold the plunger and syringe barrel together in place so that no hydrogel or PBS leaks out. Invert multiple times to mix the fragmented hydrogel with the PBS added.
Hold the syringe such that the tip cap is facing up and the plunger is facing down. Remove the tip cap. Very gently, push the plunger upwards to remove any air from the inside of the syringe.
Extrude the fragmented hydrogel solution through a series of needles to create fragmented microgels. Secure a blunt tip 18-gauge needle to the top of the syringe containing the fragmented hydrogel and PBS. Remove the plunger from a fresh 3-milliliter syringe and secure a tip cap to the empty syringe barrel.
Extrude the fragmented hydrogel solution through the 18-gauge needle into the back of the empty syringe barrel. Discard the empty syringe and needle into the proper sharps waste stream. Repeat the extruding of hydrogel solution with a 23, 27, and 30-gauge needle.
Upon the last extrusion step, extrude the fragmented microgel solution into microcentrifuge tubes. Wash and isolate the fragmented microgel suspension. Using a microcentrifuge, spin down the fragmented microgel solution at 5, 000 times g for 5 minutes.
Use a pipette to remove the supernatant. Add 1 milliliter of PBS to each microcentrifuge tube containing fragmented microgels and vortex for 5 to 10 seconds. Combine 20 microliters of fragmented microgel suspension with 180 microliters of PBS to create a dilute fragmented microgel suspension.
Vortex to mix thoroughly. Transfer 50 microliters of dilute fragmented microgel suspension to a glass microscope slide. Use an epifluorescent microscope to acquire images of fluorescently-labeled microgels at 4x or 10x zoom.
Jam fragmented microgels using vacuum-driven filtration. Assemble and test the vacuum-driven filtration apparatus. Secure a Buchner funnel inside of a filter flask.
Use tubing to connect the filter flask to a vacuum line. Place a membrane filter in the Buckner funnel cup. Turn on the vacuum line by opening the dial valve.
Test the connection by pipetting approximately 0.5 milliliters of PBS onto the membrane filter and observe that all the PBS goes through the filter and collects in the bottom of the filter flask. After turning on the vacuum line and ensuring a complete seal, vortex the fragmented microgel solution. Using a 1, 000-microliters pipette, transfer the fragmented microgel solution onto the membrane filter.
Wait for approximately 30 seconds for the vacuum to pull PBS out of the microgel solution. Turn off the vacuum line. Obtain a fresh 3-milliliters syringe and remove the plunger.
Use a metal spatula to scoop the fragmented granular hydrogel from the filter and transfer it into the back of the empty syringe barrel. Return the plunger to the syringe. Load the fragmented granular hydrogel into the syringe and it is now ready for use.
Remove the tip cap and replace it with a needle of choice. Load the syringe into the printing platform of choice. Load the prepared G-code file from the planning phase into the 3D printing software.
Navigate to the Print Preview panel and press Print. As soon as the printing deposition is complete, expose the fragmented granular hydrogel constructs to UV light for photo-crosslinking and stabilization. Extrusion fragmentation yields microgels with jagged polygon shapes with diameters ranging from 10 to 300 micrometers.
The circularity ranges from 0.2 to almost 1 and the aspect ratio ranges from 1 to 3. These parameters describe the irregular and jagged microgel shapes formed by the fragmentation process. When packed together using either centrifugation or vacuum-driven filtration, the assembled granular hydrogel is shear-thinning and self-healing.
The fragmented granular hydrogel has high-shape fidelity and mechanical integrity for an injectable hydrogel. In addition to using fragmented granular hydrogels for 3D printing, these systems can also be used as in vitro cell culture platforms, as well as injectable systems for tissue repair and drug delivery. These simple and low-cost methods bring the advantages of granular hydrogel biomaterials to more researchers allowing for increased access to granular hydrogel fabrication as well as innovation in the biomaterials field.
