Gelatin Methacryloyl Granular Hydrogel Scaffolds: High-throughput Microgel Fabrication, Lyophilization, Chemical Assembly, and 3D Bioprinting

Extracellular matrix or ECM numeric hydrogel microparticles may be used as the building blocks for scaffold fabrication. This protocol demonstrates the fabrication, purification, lyophilization, photo assembly, and 3D bioprinting of gelatin methacryloyl or GelMA hydrogel microparticles. GelMA is a photochemically, crosslinkable, protein-based biopolymer containing cell adhesive and biodegradable moieties which has been widely used as a cell-responsive and instructive biomaterial.

In comparison to conventional bulk hydrogel, GelMA granular hydrogel scaffolds are microporous, enabling rapid cell infiltration that can be tailored by the building block size. GelMA granular hydrogel scaffolds may open new opportunities for the 3D bioprinting of thick tissues, organs, and diseases on cheap platforms and regenerative engineering. To begin, add 10 milligrams of LAP to 10 milliliters of DPBS to prepare a 0.1%photoinitiator solution.

Protect the solution from light by wrapping it with aluminum foil, and label it. Next, dissolve the desired amount of GelMA in the photoinitiator solution. Place the aluminum-foil-wrapped solution in the 37-degrees-Celsius oven for one hour to get a clear solution.

To prepare the oil phase, make a 2%biocompatible surfactant solution in the engineering fluid. Insert the Tygon tubing in the inlets and outlet of the PDMS device, then insert a 25-gauge blunt needle in the other end of Tygon tubing for inlets. Place the device under the microscope, and keep the environment warm at approximately 40 degrees Celsius using a hairdryer or a space heater.

Load the aqueous and oil solutions in separate syringes connected to the device. Start the syringe pump for the oil phase with a flow rate of 160 microliters per minute. After oil fills the channel, start the aqueous phase at 80 microliters per minute.

Check the droplet formation under the microscope. Collect the droplets in a container, and evaluate them in the imaging chamber by optical microscopy imaging. Keep the droplets at four degrees Celsius overnight while protecting them from light to initiate GelMA microgel's physical crosslinking and convert the droplets to stable microgels.

To implement the microengineered emulsion-to-powder, MEtoP, method, collect the physically crosslinked microgels in the engineering fluid using thermally durable microcentrifuge tubes or cryovials. Seal the open tube with a laboratory wipe and tape. Deep-freeze the physically crosslinked microgels in liquid nitrogen for 10 minutes to proceed with lyophilization.

After lyophilization, a solid powder will be obtained. Add one milliliter of cooled, 0.1%photoinitiator solution at four degrees Celsius to the lyophilized powder to make microgel suspensions. Centrifuge the vortexed mixture at 3, 000g for 15 seconds.

After discarding the supernatant, transfer the packed microgel suspension to a mold using a positive displacement pipette, and expose it to light at 400 nanometers wavelength with an intensity of 15 milliwatts per square centimeter for 60 seconds to form granular hydrogel scaffolds or GHS. For 400 microliters of microgel suspension, add an equal amount of ice-cold PFO solution, 20%in engineering fluid to the physically crosslinked GelMA microgels. Then centrifuge the vortexed mixture at 300g for 15 seconds, and remove the oil phase from the GelMA microgels by pipetting.

Then add 400 microliters of ice-cold, 0.1%photoinitiator solution to the microgel suspension. After centrifuging the vortexed solution at 300g for 15 seconds, discard the oil. Repeat the steps of photoinitiator solution addition and centrifugation, but centrifuge at 3, 000g this time and discard the supernatant of packed GelMA microgels.

Transfer the packed GelMA microgels to a mold, followed by light exposure to form the GHS. Add 100 milligrams of nanoplatelet powder to three milliliters of ice-cold, ultra-pure water to form a 3.33%nanoparticle dispersion. Vortex the dispersion vigorously inside a four-degrees-Celsius refrigerator for 15 minutes to exfoliate the aggregated nanoparticles.

Properly exfoliated nanoparticles yield a clear dispersion. Dissolve 50 milligrams of LAP in five milliliters of ice-cold, ultra-pure water to prepare a 1%photoinitiator stock solution. Then add 333 microliters of 1%photoinitiator solution to the exfoliated nanoparticle dispersion, and wrap it in aluminum foil to protect against the ambient light.

Vortex the mixture for one minute to mix the nanoparticle dispersion and the photoinitiator. Add ice-cold PFO, 20%in engineering fluid to the physically crosslinked GelMA microgels at a one-to-one volume ratio. After centrifuging the vortexed mixture at 300g for 15 seconds, discard the oil phase containing the surfactant.

Add the ice-cold, LAP-supplemented nanoparticle dispersion to the washed GelMA microgels. After centrifuging at 3, 000g for 15 seconds, discard the remaining oil at the bottom and the supernatant dispersion. Store the suspension at four degrees Celsius while protecting it from light using aluminum foil for one day to yield the product GelMA nanoengineered granular bioink or NGB.

The following day, load the NGB into a three-milliliter syringe. Seal the loaded syringe with a cap and parafilm, and do a pulse centrifugation at 200g to remove the trapped air. Transfer the bioink to a three-milliliter cartridge using a female-female Luer lock connector.

Centrifuge the cartridge briefly at 200g again to remove the trapped air, and keep the NGB at four degrees Celsius before use. Next, load the prepared murine fibroblast cell suspension into a three-milliliter syringe. Couple the NGB and the cell-loaded syringes using a female-female Luer lock connector, and mix the contents gently by pushing back and forth 40 times.

Print the NGB or cell-laden NGB using a proper bioprinter with a standard conical nozzle by loading the nozzle into the three-milliliter printhead. Keep the printing bed temperature below 10 degrees Celsius, and optimize parameters such as speed and back pressure. Select the desirable gcode or STL file.

Then select the substrate and the nozzle type for a pneumatic three-milliliter syringe equipped with a standard conical nozzle. Calibrate the bioprinter using the device guidelines, and start printing to obtain the 3D structures. The MEtoP technology yielded resuspendable dried microgel powder by low-pressure freeze-drying while protecting microgels from aggregation and severe deformation.

The SEM images showed that the dried GelMA microparticles using this technology retain their spherical shape after lyophilization compared with the aggregation observed with conventional lyophilization. The micropore characterization of GelMA, GHS, and NGB with a high molecular weight fluorescence dye showed the interconnected microscale void spaces. Also, the fluorescence image assessed using a custom-written MATLAB script detected the pores.

The void fraction and median pore equivalent diameter measurements showed no significant difference attesting to the availability and interconnectivity of microscale pores in the 3D-printed scaffolds using the NGB. The superiority of NGB scaffolds to tightly and loosely packed GelMA microgels by the hanging filament length measurement revealed that NGB had a greater length than the packed microgels. The loosely packed microgels did not yield filaments.

A hollow cylinder was 3D-printed, and the whole construct was exposed to light for photocrosslinking. The printed structure was physically held to show the shape, fidelity, and structural integrity. If the scaffold is used for biological assessment, for example, in cell-laden bioprinting, remember to perform all the steps under certain conditions, under the biological safety cabinet.

We hope to democratize the use of granular hydrogel scaffolds as a newly emerged biomaterial platform in biomedicine.