To begin 3D printing of the phototunable hydrogel constructs, place the glass syringe with the hydrogel bio-ink 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 needle's 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 phototunable hydrogel bio-ink. 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 bio-ink 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.
