The fabrication of tubular structures is a promising approach to tissue engineering of vascular networks, which remains one of the major challenges in cultivation of thick tissues in-vitro. The main advantage of the core/shell technique is the simple production of hollow filament scaffolds in a single step, reducing the fabrication time and potentially allowing direct printing with cells. Preparing a hydrogel formulation with the appropriate viscoelastic properties, and sustaining a continuous balanced flow of the core/shell materials is crucial for 3D printing of stable scaffolds.
To begin, fill the sterile 5 mL syringe with freshly prepared hydrogel. Fill a second 5 mL syringe, equipped with a 27 gauge blunt-end needle, with freshly prepared crosslinking solution. Assemble the core/shell nozzle.
Insert a G27 blunt-end needle as the core component and fasten the needle using the screw. The inner needle should protrude about 1 mm from the outer core shell nozzle. Connect the syringe with the crosslinking solution to G27 needle in the nozzle and load the syringe into one of the extruder mounts of an ethanol-sterilized 3D printer.
Mount the syringe with hydrogel into the second extruder and connect it to the nozzle. Use a Luer lock to connect a short tube to the side Luer input of the core/shell nozzle. Carefully manually adjust the alignment until the nozzle touches the surface before retracting the nozzle to a distance equal to the outer nozzle diameter.
Import the generated scaffold g-code and press Play to initiate the printing process. After printing, carefully remove the substrate with the printed scaffold and pour the secondary crosslinking solution over the entire scaffold. Incubate the scaffold for one minute at room temperature.
To detach the scaffold from the substrate, gentle pull the scaffold sideways. If the scaffold adheres strongly to the substrate, insert a sharp edge between both materials to separate them. Transfer the scaffold to a fresh container.
UV sterilize both sides of the scaffold for 30 minutes per side. Then place the scaffold into a colorless cell culture medium and incubate the scaffold at 37 degrees Celsius with 5%carbon dioxide for at least 24 hours. The next day, harvest huvex from an in-vitro culture with 0.25%trypsin for five minutes at 37 degrees Celsius.
When the cells have detached, stop the enzymatic reaction with 3 mL of fresh cell culture medium and collect the cells by centrifugation. Re-suspend the pellet in fresh culture medium containing phenol red, and dilute the cells to a 3.4 times 10 to the fifth endothelial cells per milliliter concentration. Load the cells into a sterile 5 mL syringe equipped with a 27 gauge needle and locate an entry point in the scaffold.
Aligning the needle with the straight section of the scaffold filament, carefully puncture the scaffold at a low angle. Confirm that the needle is within the hollow filament of the channel and gently depress the plunger to inject approximately 1-2 mL of cell suspension into the scaffold. The flow of the suspension should be visible through the translucent scaffold.
When the entire scaffold has been filled with cell suspension, submerge the scaffold in fresh cell culture medium, and return the scaffold to the cell culture incubator for up to 10 days. For live/dead imaging at the end of the incubation, rinse the scaffold with PBS, and use a blunt-end needle to carefully inject live/dead solution into the scaffold. Confirm that the solution has filled the entire scaffold, and place the scaffold at 37 degrees Celsius for 30 minutes.
At the end of the incubation, rinse the scaffold with PBS, and carefully transfer the scaffold to a glass slide. The dyed cells can then be observed in the scaffolds under a fluorescence microscope. In this cross-section of a freshly printed and post-processed scaffold, a clearly visible hollow channel can be observed within the filament.
Even after 72 hours of incubation in cell culture medium at 37 degrees Celsius as demonstrated, the filament retains the hollow structure throughout the entire scaffold length. Here, a representative live/dead assay of endothelial cells after 48 hours of culture within a scaffold is shown. The live cells can be identified by their bright green fluorescent signal.
This technique is the basis for the one-step fabrication of hollow tubular scaffolds and can be upgraded by direct printing using cells incorporated into the bio-ink.
