A microscope image of a 3-D printed sugar template used for creating vasculature in living tissues. (Photo by Jordan S. Miller)

Scientists are able to make 2-D structures out of many types of tissues. However, creating 3-D structures is more difficult because blood vessels are required to prevent cells from suffocating. To overcome the challenge of fabricating blood vessels in 3-D engineered tissues, researchers at the University of Pennsylvania (Philadelphia) and the Massachusetts Institute of Technology (MIT; Cambridge) have developed a method for printing 3-D vasculature that relies on the use of sugars.

The most commonly explored technique for making vasculature is layer-by-layer fabrication, or bioprinting, in which single layers or droplets of cells and gel are created and then assembled together one drop at a time. While such additive manufacturing methods can make complex shapes out of a variety of materials, creating vasculature remains a major challenge when printing with cells. Hollow channels made using this method have structural seams running between the layers, and the pressure of fluid pumping through them can push the seams apart. More important, many potentially useful cell types, such as liver cells, cannot readily survive the rigors of direct 3-D bioprinting.

To surmount this obstacle, Jordan S. Miller and Christopher S. Chen at Penn turned the printing process inside out. Rather than trying to print a large volume of tissue and leave hollow channels for vasculature in a layer-by-layer approach, they focused on the vasculature first and designed free-standing 3-D filament networks in the shape of a vascular system that sat inside a mold. As in lost-wax casting, the team’s approach allowed for the mold and vascular template to be removed once the cells were added and formed a solid tissue enveloping the filaments.

However, this rapid-casting technique depended on the researchers developing a material that is rigid enough to exist as a 3-D network of cylindrical filaments but which can also easily dissolve in water without toxic effects on cells. The material also had to be compatible with a 3-D printer to accelerate the process of making reproducible vascular networks that are larger and more complex than those that can be fabricated using a bioprinting approach. After much testing, the team hit on the use of sugar. Mechanically strong, sugars form the majority of organic biomass on the planet in the form of cellulose, but their building blocks are also typically added and dissolved into nutrient media that help cells grow.

Using a combination of sucrose and glucose, along with dextran for structural reinforcement, the researchers printed vasculature using a 3-D printer. To stabilize the sugar templates after printing, the scientists then coated the templates with a thin layer of a degradable polymer derived from corn, a process that enables the sugar template to be dissolved and to flow out of the gel through the channels they create without inhibiting the solidification of the gel or damaging the growing cells nearby. Once the sugar was removed, the researchers flowed fluid through the vascular architecture to provide the cells with nutrients and oxygen, a process that is similar to the exchange that naturally occurs in the body.

The researchers showed that human blood vessel cells injected throughout the vascular networks spontaneously generated new capillary sprouts to increase the network’s reach, replicating how blood vessels grow in the body. The team then created gels containing primary liver cells to test whether their technique could improve the cells' function. When the researchers pumped nutrient-rich media through the gel’s template-fashioned vascular system, the entrapped liver cells boosted their production of albumin and urea, which are important measures of liver-cell function and health. There was also clear evidence of increased cell survival around the perfused vascular channels.

“The therapeutic window for human-liver therapy is estimated at one to 10 billion functional liver cells,” remarks MIT's Sangeeta N. Bhatia. “With this work, we’ve brought engineered liver tissues orders of magnitude closer to that goal, but at tens of millions of liver cells per gel, we’ve still got a ways to go."