Engineers have developed a new process of 3D bioprinting tissues that uses multiple cell-based inks to create more realistic structures in less time than previous methods.
Engineers have developed a new process of 3D bioprinting tissues to create more realistic structures in less time than previous methods. The approach integrates a microfluidic system to deliver four cell-based inks to a stereolithographic 3D printer that sets the material using UV light.
The system, according to the researchers, provides a robust platform for bioprinting high-ﬁdelity multimaterial microstructures on demand for applications in tissue engineering, regenerative medicine, and biosensing. Conventional stereolithographic biofabrication platforms print those structures too slowly for practical purposes.
In tissue engineering, 3D printers are useful tools because they create structures with shape, volume, and porosity that resemble natural tissues. But most 3D printers only print one type of cell-based ink at a time. Manually swapping out inks to print different cell types in specific locations, as in heart, liver, and bone, is time consuming.
A team led by Dr. Yu Shrike Zhang, a bioengineering instructor at Harvard University, and Dr. Ali Khademhosseini, a bioengineering professor at the University of California, Los Angeles, wanted to print multiple cell-based inks quickly and precisely. As reported in Advanced Materials, the researchers developed a microfluidic system to deliver one of four cell-laden polymer inks into a central chamber.
They positioned this chamber underneath the nozzle of a stereolithographic 3D printer. A pre-programmed pattern of UV light shines through the nozzle, triggering a chemical reaction in the liquid ink. This reaction forms crosslinks between polymer chains, causing the material to solidify.
The pattern comes from a 1050 x 920 array of micromirrors that are digitally programmed to be dark or light-reflecting. These mirrors have a 10 kHz switching speed, meaning that new patterns can be produced quickly as different bioinks flow into the central chamber for a few seconds. The team printed structures made from two or three different bioinks in 20 seconds, compared to minutes needed to create the same structure by manually changing inks on an industrial stereolithographic 3D printer.
Stereolithographic bioprinting generally produces smaller features than other types of 3D bioprinting. With this system, the researchers could print parallel lines as thin as 25 µm, as well as eccentric circles located one inside the other each with different center points. They also used multiple colored bioinks to print 3D structures, such as a star-shaped pyramid.
The team wanted to integrate the microfluidics and 3D printing as much as possible so they could build reliable and reproducible scaffolds. But that integration was more difficult than the team hoped, Khademhosseini says. Synchronizing ink delivery through the microfluidics with the movement of the printer nozzle where the light comes through was challenging, he adds.
Printing multiple materials also adds challenges beyond the issue of keeping cells alive during the printing process. Mismatches in the chemical or mechanical properties of different materials printed in the same scaffold could cause layers to separate.
Finally, the researchers used their system to print biologically relevant structures, such as a model of muscle tissue and the tendon-bone interface. To recreate the tissue bundles in muscle, the researchers printed overlapping squiggles of gelatin methacryloyl (GelMA) inks laden with skeletal muscle cells or fibroblasts, which are cells in connective tissue that produce collagen.
The researchers used three GelMA inks to recreate the tendon-bone interface. They printed a pool of an osteoblast-containing ink. Osteoblasts are cells in bone that produce minerals. They dotted the pool with spots of an ink containing human mesynchymal stem cells found in bone marrow. Narrow strips of polymer containing fibroblasts stretched from this pool to mimic tendon.
In the future, the researchers imagine modifying the microfluidic system to add as many bioinks as needed to print a desired structure. However, the size of the structures they can currently produce is limited by the size of the microfluidic chamber. Scaling up this method will require altering the chamber, the researchers say.
Melissae Fellet is an independent technology writer.
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