3D Bioprinting Phototunable Hydrogels to Study Fibroblast Activation

We engineered 3D models of lung tissue to help the world breathe easier. Our mission is to use 3D biomaterials and techniques like 3D printing to build platforms that help us understand what is causing chronic lung diseases and how to better treat these conditions. One experimental challenge in our research is the difficulty of 3D printing soft materials into complex shapes.

We precisely deposit cells by printing our bioink into a shear-thinning support bath that helps maintain their shape during the printing process. Our biomaterials provide control over sample mechanical properties throughout the entire course of the experiment. This formulation allows researchers to grow cells in a hydrogel that matches the stiffness of both healthy and diseased lung tissue.

Users can decide when to stiffen the samples and measure cellular responses. The phototuneable biomaterials used in this protocol allow for a softened model to be created, which can later be stiffened to explore cellular responses to microenvironmental stiffening. Similar techniques that use a static biomaterial instead of a phototuneable biomaterial result in a model that cannot be stiffened after printing.

Results from other studies in our lab suggest sex and age play critical roles in lung disease progression. Future models will study these variables and include more complexity like additional cell-specific layers. Coupling these efforts will result in more realistic bioprints and lead to new ways of treating fibrotic diseases.

Begin the preparation of the hydrogel bioink aseptically inside a biosafety cabinet by first preparing a 0.25 milligrams per milliliter stock solution of PEG alpha methacrylate in sterile cell culture media without FBS. Also, using 20 millimolar sterile TCEP, prepare 250 millimolar stock solutions of DTT MMP2 degradable crosslinker and the RGD peptide. Finally, prepare a 15 weight percent stock solution of PEO using distilled water.

Combine the required amounts of the prepared reagent stock solutions and low-serum cell culture media with the fibroblasts in a 15 milliliter conical tube. Mix the resultant bioink thoroughly using a positive displacement pipette to ensure the cells are single. Verify that the final precursor solution has a pH of 6.2.

Remove the printhead from the bio-printer to access the glass syringe. To load the bioink into the glass syringe remove the syringe plunger, then collect the bioink from the centrifuge tube using a separate syringe fitted to a 1.5 inch long 15 gauge blunt tip needle. Replace the 15 gauge needle with a 30 gauge 0.5 inch long blunt tip needle and transfer it to the glass syringe, avoiding air bubble formation.

Finally, place the glass syringe within the printhead and attach the printhead components while ensuring a firm assembly for printing. To begin 3D printing of the phototuneable hydrogel constructs, place the glass syringe with the hydrogel bioink containing fibroblasts within the printhead and attach the printhead components while ensuring a firm assembly for printing. Then using the directional arrows in the Pronterface software manually and carefully adjust the extrusion needles position within the support bath slurry in the center of the well of the well plate.

Leave at least one millimeter of slurry below the needle tip. Once the needle tip is correctly placed, press the print button in the Pronterface software and wait for the printing to complete. Repeat the previously demonstrated steps until the desired number of bioprinted constructs are obtained.

Following printing, leave the well plate with the constructs covered at room temperature in the biosafety cabinet for one hour. This allows for base-catalyzed polymerization to occur with the phototuneable hydrogel bioink. Then place the well plate with the 3D bioprinted constructs in a 37 degree Celsius sterile incubator for 12 to 18 hours to melt the support bath slurry.

Next, inside a biosafety cabinet, change the media surrounding the bioprinted constructs. To do so, manually remove the media and the melted gelatin support bath from the wells without disturbing the constructs. Then add an appropriate volume of low serum media to each well.

Again, 24 hours prior to the desired stiffening time point replace media in the wells with low serum media supplemented with 2.2 millimolar sterile LAP. At the desired stiffening time point, remove half of the media from the wells to be stiffened and place the plate without the lid under the ultraviolet or UV light of an OmniCure. Turn on the UV light with a 365 nanometer band pass filter to stiffen the constructs for five minutes.

Once stiffened, remove the remaining media from these wells before adding fresh low serum media to each well. Return the plate to the incubator until performing the fibroblast activation study at the desired time point. The combination of an acidic bioink and basic support bath slurry facilitated the polymerization of the 3D bioprinted constructs and maintained the cylindrical structures.

Microscopy of fluorescently labeled hydrogel showed pores within the hydrogel induced by gelatin microparticles in the support bath. Confocal microscopy showed spatial control over stiffening in 3D. Fibroblast viability assays revealed that constructs with 300 micron wall thickness and having 4 million cells per milliliter outperformed all other conditions at every time point.

Viability peaked on day seven with about 91%of the cells staining live, and by day 14, 85%were still viable.