The overall goal of this protocol is to demonstrate a versatile approach for designing hydrogel bioinks that can be extruded through bioprinting devices. The bioinks can then be used to fabricate three dimensional tissue constructs. This method can help answer key questions in the bioprinting field, such as how to control the mechanical properties needed in order to provide a material that can be extruded using a bioprinter.

The main advantage of this technique is that we use commercially available components combined in a modular fashion to create a simple and effective bioprintable hydrogel bioink. The applications of these technologies include the creation of 3D tissue organoids that can be used to accurately model the effects of drugs, toxins, and diseases. Although this method can provide a framework to bioprint 3D liver constructs, it can also be applied to other tissue types such as muscle, lung, and colon.

Generally, individuals new to this method will struggle because there are a number of different reagents used to create the hydrogel bioink, but it is actually quite straightforward. Demonstrating the procedure will be Young-Joon Seol, a post-doc on our team. To begin, prepare a tissue-specific extracellular matrix digest to be used in the hydrogel formulation as described elsewhere.

Then, dissolve a photoinitiator in water at a weight per volume ratio of 0.1%To form the hydrogel bioinks, first dissolve the base material components from the hyaluronic acid hydrogel kits into individual aliquots of the water photoinitiator solution. Then, combine the ECM solution, the 2%thiolated hyaluronic acid, the 2%thiolated gelatin, the crosslinkers, and hepatocyte culture media in the ratios shown here. To improve the extrusion properties of the bioink, add 1.5 milligrams per milliliter of unmodified hyaluronic acid and 30 milligrams per milliliter of gelatin to the mixture.

Then, vortex the resulting mixture on high for ten seconds prior to use. Before testing the bioink in a bioprinting device, first test the extrusion characteristics on the laboratory bench. Using a standard syringe, drop a sample of the bioink and then attach a 20 to 30 gauge needle to the syringe.

Allow the bioink to crosslink and then push the bioink through the needle to achieve smoothly extruded hydrogel filaments. If the formulation is able to create a filament with few or no bumps, then it is ready for bioprinting. To load the bioink preparations in a bioprinter, pipette the bioinks into sterilized printer cartridges.

Allow them to sit for 30 minutes before extrusion as the bioink will undergo spontaneous stage one crosslinking within the cartridge. Next, load the cartridge into the print setup and connect a pneumatic pressure source to the cartridge. Prepare a simple pattern, like this seven by seven millimeter grid of parallel lines, to print in order to evaluate its extrusion compatibility.

While the print head moves in the XY plane at a velocity of approximately 300 millimeters per minute, apply a pressure of 20 kilopascals to the cartridge to extrude the bioink. If the extruded materials are lumpy or irregular, reduce the amount of the crosslinker added in order to soften the stage one crosslinked material. A properly prepared bioink formulation should extrude smoothly, allowing for precise deposition and architectures.

Prepare 3D primary cell liver spheroids in a 96 well plate using the hanging drop method as described in the accompanying text protocol. After three days in culture, collect the liver spheroids from the hanging drop plate using a pipette, and transfer them into a sterile, 15 milliliter conical tube. Let the spheroids settle to the bottom of the conical tube for one to two minutes.

Then, carefully aspirate the media with a pipette. Transfer 110 to 125 percent of the desired printed 3D construct volume of the freshly prepared hydrogel bioink solution to the conical tube containing the spheroids. Then, carefully pipette the spheroids up and down to re-suspend them in the hydrogel bioink solution.

Once evenly suspended, transfer the spheroid solution to a bioprinter cartridge using a pipette and allow the solution to undergo the first crosslinking stage for 30 minutes. Following stage on crosslinking, use a bioprinting device to create the desired hydrogel structures containing the primary liver spheroids. After each layer of deposition, expose the printed bioink to UV light for two to four seconds to initiate the secondary crosslinking mechanism.

This will stabilize the constructs and increase the stiffness to the desired level. The concentration of PEG alkyne in the solution is what controls the overall crosslinking density, and therefore primarily controls the stiffness of the final construct. After bioprinting, high cell viability in the liver constructs was observed using confocal microscopy.

Under optimal environmental conditions, viability should be above 85%Additionally, when the constructs were stained for markers indicative of liver tissue, positive expression of CYP3A4, a cytochrome P450 isoform, intracellular albumin, E-cadherin, an epithelial cell-cell adhesion protein, and DPP4, a protein expressed highly in the liver, were all observed. When the culture media was tested for levels of urea and albumin, it was found that the construct secreted both urea and albumin at constant levels over the 14 day time course. This further suggests that the tissue-specific hydrogel bioinks aid in maintaining the function of the liver cells.

Once mastered, this technique can be done in about two hours from start to finish if it is performed properly. However, this is often dependent on the particular bioprinting device that is employed. While attempting this procedure, it's important to remember that the steps demonstrated must often be adapted to be compatible with other tissue types or bioprinting devices.

Following this procedure, other hydrogel bioink formulations can be created in order to support the bioprinting of other tissue types. The development of these technologies helped pave the way for the creation of multi-organoid, body-on-a-chip platforms for the screening of drugs and modeling diseases. After watching this video, you should have a good understanding of how to begin designing materials that can be used for bioprinting 3D tissue constructs by using multi-step crosslinking.

Don't forget that working with ultraviolet light can be extremely hazardous to one's vision, and precautions such as using UV protective goggles should always be taken while performing this procedure.