Microgel-Extracellular Matrix Composite Support for the Embedded 3D Printing of Human Neural Constructs

Embedded 3D Printing within SHAPE composites overcomes traditional biofabrication limitations to enable accurate patterning of mechanically-sensitive tissues inside hydrogels that resemble the native extracellular environment. SHAPE toolbox relies on modular biomaterial design, allowing easy changes in printing support formulation. It also leverages low cost materials and accessible equipment for simple adaptation by other research groups.

This approach can be applied to create spatially-defined human neural tissue models for examining neuronal development and communication in neurodegenerative disorders such as Parkinson's. Begin by preparing calcium carbonate solution in ultrapure water at two milligrams per milliliter concentration. Mix it with the alginate solution at an equal ratio and magnetically stir it at 650 RPM for one hour at room temperature.

Then add acetic acid to this mixture at a ratio of one to 500 and stir again overnight. The next day, mechanically fragment the jelled alginate solution into microparticles at 15, 000 RPM for 10 minutes using a homogenizer. Then centrifuge to obtain the microparticles.

Carefully discard the supernatant and resuspend the particles in the DMEM, containing two millimolar sodium hydroxide and 1%penicillin streptomycin. The color of the suspension should change back to red. Incubate the suspension overnight at four degrees Celsius.

After incubation, homogenize the suspension for three minutes at 15, 000 RPM and centrifuge at 18, 500 G for 10 minutes. After centrifuging, remove the supernatant and observe the pellet for tightly-packed alginate microparticles. To generate the SHAPE composite, mix the alginate microparticles pellet with diluted and neutralized collagen at a ratio of two to one and pipette it up and down on ice.

Transfer the generated composite material into a cooled 24 well plate. Before printing, dissociate the previously cultured human neural stem cells or hNSCs using a 0.025%trypsin solution for five minutes at 37 degrees Celsius. Then neutralize the trypsin with the growth medium and centrifuge the cell suspension at 400 G for five minutes at room temperature.

After removing the supernatant, resuspend the cell pellet in two to three milliliters of growth medium. Using a 21 gauge blunt metal needle, load 100 microliters of tightly-packed alginate microparticles into the syringe. Then load the prepared cell suspension into a syringe.

For printing, replace the loading needle with a 27 gauge blunt metal needle. Using referenced software, once the structure to print is designed, click on generate for a G code generation. Insert the cell loaded syringe into a volumetric extrusion head on an extrusion-based bioprinter and record the needle length by clicking on start needle length measuring process.

Then place the 24 well plate loaded with SHAPE composite onto the printer, and click on SHM to measure the empty well surface height. Select pH 2 followed by wrench symbol. Then under the parameter set, change the volume flow rate to 3.6 microliters per second and press Apply Parameter, followed by Save and Done.

Load the G code into the printer user interface by clicking an icon for open G code. Select the icon for the run program to initiate the print procedure. Immediately after printing, incubate the SHAPE gel at 37 degrees Celsius for 30 minutes for annealing.

Then gently add growth medium to the annealed SHAPE gel support. For immunostaining, remove the excess medium from the gel, and using a spatula, transfer the gel to a container containing DPBS. Once the plate is placed in the fume hood, remove the DPBS.

Cover the gel with 4%formaldehyde solution and incubate for one hour at room temperature. After washing with DPBS, add the blocking solution to the gel. Rock the plate gently and incubate for six hours at room temperature to prevent unspecific binding.

Once the blocking solution is removed, add the primary antibody to the gel and gently rock it. Incubate the plate for 48 hours at four degrees Celsius. After DPBS wash, similarly treat the gel with the secondary antibody solution for 24 hours.

Place the stained gel with a spatula into a well plate with a thin imaging bottom before imaging. In the present study, the hNSC ink printed a filament of cells with a diameter of around 200 micrometers. The printed strands were rich with viable cells with around morphology.

After 30 days of printing, the cells exhibited neural morphology with small cell bodies and long thin processes, which indicated the successful differentiation of hNSCs. Staining with tubulin beta three allowed visualization of the generated neural networks with maintained geometry. And during the differentiation process, the cells did not migrate out of the printed strands.

SHAPE composite can be used to produce perfusible channels by printing sacrificial ink instead of cells. Moreover, the support can include oxygen sensitive beads to monitor oxygen tension in 3D printed structures over time and space.