Agarose Fluid Gels Formed by Shear Processing During Gelation for Suspended 3D Bioprinting

The ability to use different bioinks is paramount to the successful development of bioprinted structures, and this technology facilitates the use of materials that are generally incompatible with conventional printing techniques. The suspending medium prevents the collapse of low viscosity bioinks when deposited, and allows the integration of different bioinks within a single construct to create regional variations in chemical and mechanical properties that are more akin to native tissue. Demonstrating the procedure will be Dr.Tom Robertson, a research fellow from the University of Birmingham, and Dr.Jessica Senior, a research fellow from the University of Huddersfield.

To begin, turn on the rheometer and insert 40 millimeter serrated geometries, allowing it to stand for 30 minutes. Zero the gap height of the rheometer using the zero-gap height function. Add approximately two milliliters of sample on the bottom plate and lower the top geometry to create a gap height of one millimeter.

Trim the sample by removing excess material expelled from between the plates using a flat, non-abrasive edge to pull excess fluid away from the gap, and soak it up with tissue paper. To determine the injectability of the bioink, undertake viscometry profiles by selecting Viscometry test from the user options. Input the parameters for a shear rate controlled ramp test at 0.1 to 500 per second with a one-minute ramp time.

Load a new sample and repeat the process to ensure reproducibility. To determine the gelling characteristics of the bioink, small deformation tests were performed by selecting Oscillatory testing from the user options. Input parameters into a single frequency test under constant strain, as the frequency of one hertz, strain of 0.5%over one hour while the ink gels.

Repeat the viscometry ramp test on new samples under stress control using the upper and lower stresses determined from the shear rate controlled ramp test in step as demonstrated previously. To perform in situ amplitude and frequency measurements on gelled samples, select an Oscillatory test from the user options. After selecting the amplitude sweep, input the parameters for an amplitude sweep test that is strain controlled at 0.01 to 500%at a constant one hertz frequency.

After completing the test, analyze the spectra to determine the linear viscoelastic region. Load a new sample on the rheometer and allow it to gel. Then select an Oscillatory test from the user options.

Select frequency test and input frequency parameters between 0.01 and 10 hertz and a strain within the linear viscoelastic region of the spectra determined from the amplitude sweep data obtained previously. To launch CAD software and to start the generation of a CAD model, select the Tools option, and then click on Materials in the CAD software to define the printing parameters for the chosen bioink. Input the estimated filament diameter in the Thickness tab to determine the z-thickness of each layer.

To design the structure layer by layer, use the Layer tabs in the software, group the layers using the Group tab, and assign each layer to a level on the z-plane using the Level tab. Generate a lattice structure by creating one layer with the filaments along the x-axis and a second layer along the y-axis and assign both to a separate level. Under the Group tab, determine the build height by selecting the number of repeated units in the structure.

Then click the Generate tool to create a G-code for the design and view a 3D render of the structure. In the bioprinter, aliquot the bioink into the printing cartridge, and screw it into the print head above the micro-valve. Then click the needle length measurement function to calibrate the print head.

Next, connect the assembled print head to the pneumatic pressure system, and load the culture vessel onto the printing platform. Once an appropriate pressure has been selected, open the G-code generated previously, and click Run to initiate the printing process. Once the printing of the carotid artery is complete, use a syringe and needle to inject two milliliters of 200 millimolar calcium chloride dihydrate around the construct.

After a minimum of three hours, remove the fluid gel support bed from around the construct and gently wash it in PBS. Then remove the construct from the support bath using a spatula. Tailoring print resolution of alginate and collagen lattices as a function of filament diameter via extrusion at 30, 60, and 120 kilopascal was observed and the results demonstrated that the resolution was directly tuneable with the changes in extrusion pressure.

To achieve a gradient of mechanical properties similar to those found in the skin, different proportions of pectin and collagen were used in the dermal and hypodermal layers, resulting in a structure with no sign of delamination. A high level of cell viability was observed throughout the structure following 14 days of culture during which the materials stiffened, indicating remodeling of the material. For the rheology, it's important that the sample is loaded correctly to ensure reproducible data, and for the bioprinting, it's important that the structure is fully cross-linked prior to removing it from the supporting bed.

Culturing large scale tissue constructs for extended periods helps explore the embedded cells'response to physical and chemical stimuli.