Using Multilayered Hydrogel Bioink in Three-Dimensional Bioprinting for Homogeneous Cell Distribution

Using our protocol, the multilayering of the liquid-like bioink dilutions does not only promote a homogeneous dispersity of the encapsulated cells but also maintains a cell-suitable bioink environment. We use the interfacial tension to stop the sedimentation of the encapsulated cells within the bioink reservoir, avoiding the high shear stress load to the cells during extrusion printing. Using this technology, we can generate 3D tissue constructs with a homogeneous cell distribution, providing a functional in vitro tissue mold for use in drug screening and regeneration medicine studies.

When preparing the bioink, it is important to pay attention to the experimental temperature and the hygrogel loading procedure, as these factors can affect the quality of the ink. Before beginning the preparation, use 0.22-micrometer syringe filter units to sterilize all of the materials, and dissolve gelatin to a 10%final concentration in 50-degrees-Celsius PBS in a biological safety cabinet. Add methacrylic anhydride to the gelatin solution at a 0.6 to one weight ratio with slow stirring for at least one hour at 50 degrees Celsius.

At the end of the incubation, transfer the mixed solution into a sterile 50-milliliter tube, and separate the solution into layers by centrifugation. Collect the upper GelMA layer, and dilute the collected solution with two volumes of 40 to 50-degrees-Celsius deionized water. Dialyze the GelMA solution with a 12 to 14-kilodalton molecular weight cutoff dialysis membrane against deionized water for five to seven days at 40 to 50 degrees Celsius, changing the water twice daily.

At the end of the dialysis, freeze the GelMA solution at minus 80 degrees Celsius overnight. Next lyophilize the GelMA solution for three to five days in a freeze dryer at minus 45 degrees Celsius and 0.2 millibars of pressure. Then dissolve the lyophilized GelMA in PBS supplemented with 10%fetal bovine serum, 25 millimolar HEPES, and 0.5%photoinitiator.

To obtain the silk fibroin GelMA bioinks, mix the GelMA solution with different volumes of initial silk fibroin solution and different volumes of PBS as suggested in the table. When all of the bioinks have been prepared, sonicate the solution for 10 to 20 minutes. While the inks are sonicating, collect the cells from an 80%confluent NIH/3T3 cell culture into one 15-milliliter conical tube per bioink solution, and pellet the cells by centrifugation.

Then carefully aspirate the supernatants and resuspend the cells in each tube with two milliliters of bioink at a one times 10 to the six cells per milliliter of bioink solution concentration. To load the bioink for printing, add aspirate 400 microliters of cell-laden silk fibroin 0.5 GelMA to the bottom layer of a two-milliliter syringe. Place the syringe in a zero-degrees-Celsius ice water bath for five minutes to transform the bottom layer bioink into a gel state before loading 400 microliters of cell-laden silk fibroin 0.75 GelMA bioink over the layer of silk fibroin 0.5 GelMA.

Then return the syringe to the ice bath for five minutes, continuing to add each additional concentration of cell-laden bioink to the syringe in the same manner. When a multilayered bioink system with different concentrations of silk fibroin within the different layers has been achieved, reheat the bioink in a 37-degrees-Celsius incubator for 30 minutes. At the end of the incubation, load a 27-gauge printing nozzle onto the syringe, and set the flow speed to 50 microliters per minute, the moving speed of the nozzle to two milliliters per second, and the height of the nozzle to one millimeter.

Next print the tissue construct in an extrusion manner using a custom-made bioprinter at room temperature, and cross-link the bioprinted tissue construct with 365 nanometers of ultraviolet light for 40 seconds at 800 milliwatts. Then culture the cross-linked tissue construct in DMEM supplemented with 10%fetal bovine serum and 1%penicillin-streptomycin in a cell culture incubator, changing the medium every eight hours during the first two days and every two to three days thereafter. In this representative analysis, as the bioprinting procedure progressed, the density of cells in the target wells decreased in all of the groups.

In the silk fibroin zero GelMA group, approximately 70%of the cells were deposited in the bottom layer. In the silk fibroin one GelMA group, approximately 40%of the cells were deposited in the bottom layer, and approximately 5%of the cells were deposited in the top layer. In the silk fibroin multilayer GelMA group, the deposition of encapsulated cells was more homogenous than that of the control groups.

The most critical part of this protocol is the loading of the SF-GelMA bioink multilayers. This protocol may be applied to other extrusion bioprinting analysis that use liquid-like bioinks.