Ali Khademhosseini constructed his first “living Legos” in 2006, each one a string of synchronously beating cardiac muscle cells. Similar to Legos, these strings link together to create a larger structure, in this case, pulsing sheets of engineered heart muscle. These living building blocks were evidence that a bottom-up approach to tissue engineering might just work.
Tissue engineering has traditionally relied on a top-down, scaffold-based approach. Though this method has produced some success stories, such as engineered skin, it has fallen short of recreating the cellular structures in organs such as the liver and heart. By adding a novel toolkit to the engineer’s arsenal, Khademhosseini’s work may help solve this problem. In fact, it will likely be a combination of approaches that will yield functional, marketable engineered organs.
To that end, Khademhosseini, an HMS assistant professor of medicine at Brigham and Women’s Hospital who is trained in chemical and biological engineering, and his multidisciplinary team are looking for streamlined ways to build tissue constructs. These products will have to mimic the physical, chemical, and biological interactions in natural tissues at different scales, from molecules to cells to building blocks to organized clusters.
After building his first heart strings, Khademhosseini worked on becoming deft at making a variety of living building blocks. At the nanoscale, he “played with the chemistry and materials,” infusing the cell-laden gels with extracellular matrix molecules and gradients of signaling molecules to influence cell development and aggregation, he said. At microscales, he employed fabrication technologies borrowed from the semiconductor industry, such as photolithography, to mass-produce identical building blocks in a variety of shapes and sizes.
Having developed a knack for making the blocks, Khademhosseini turned his attention to assembling them into larger structures. More than that, his team recognized that these engineered organs must be easy to assemble en masse if they are going to break out of the laboratory and become available to the millions of patients who need them. Their most recent advancement, self-assembling living Legos, represents a novel approach to reaching that goal.
Tissue engineers have traditionally employed cellular scaffolds to try to recreate the geometric constructs found in natural tissue, such as the fibrous muscles in the heart or the hexagonal lobules in the liver. This approach treats individual cells as “smart bricks” that when put in the right places and given the right cues will develop into the desired structures. “For 25 years we have relied on the inherent smartness of those bricks,” said Joseph Vacanti, the John Homans professor of surgery at HMS and Massachusetts General Hospital. “This can work well or not so well.”
“The proper geometry is often difficult to achieve,” said Khademhosseini. “In a tissue-engineered liver, for example, you might get some aggregates of hepatocytes, but you never fully recreate the lobule architecture.”
Khademhosseini wanted to solve this problem with his building blocks, which can contain hundreds of cells each, as prefabricated modular components of an organized tissue. He also sought to automate the assembly of these components.
“We want to have a system that can be mass-produced,” said Khademhosseini. “If we can make building blocks, but we have to manually put them together, then that’s not really a scalable technique.”
“Ali is thinking all the way through to what the real goal is, which is to improve patient care,” said Vacanti. This kind of thinking helps avoid hitting a “dead end, not because the science or engineering doesn’t work, but because it can’t be scaled to human use.”
In their first attempt at self-assembly, Khademhosseini and his colleagues, co–first authors Yanan Du, a Harvard–MIT Health Sciences and Technology research affiliate, and Edward Lo, an undergraduate at the University of Toronto, took advantage of a familiar fact: oil and water don’t mix; rather, the two substances separate from one another, minimizing contact. The team suspected that their hydrophilic building blocks might behave in the same way if immersed in oil.
To test the idea, they created a grid of cubic hydrogels, scraped them into a dish of mineral oil and dragged a pipette tip across the mound, breaking it up and agitating it. Depending on how fast they stirred, how long, and the composition of the oil, the blocks assembled into different shapes, such as long chains, random blobs, and offset stacks. The researchers also directed the assembly of interlocking shapes, such as stars and rods that fit together like puzzle pieces.
These techniques, described in the July 15 Proceedings of the National Academy of Sciences, could someday be used to mass-produce tissue constructs that mimic natural tissue from the bottom up.
“These microgels have an intrinsic structure and a superstructure realized by this clever assembly process he has developed,” said Jeff Borenstein, director of the biomedical engineering center at Draper Laboratory. “Together those create the mechanical properties, the biochemical properties, and the cellular microenvironment needed to move towards a functional engineered organ. This is a big step forward because it is replicating more of the features of the target organ than previous approaches.”
But “we are still at the early stages,” cautioned Khademhosseini, who is continuing to refine the approach.
On one front, his lab is working on improving the viability of the cells trapped inside each building block. To develop the self-assembly techniques, the team used a polymer that gels under ultraviolet light, which may damage cells. They are now experimenting with other materials, such as collagen, that gel using heat or other types of light.
On another front, the team is working to scale from assembling a handful of blocks to making assemblies of assemblies that form larger, millimeter- to centimeter-sized constructs. They also are experimenting with shapes besides rods and stars, such as a donut shape that “we can assemble into tubelike structures to make blood vessels,” said Du. Infusing engineered tissues with an effective vascular system has been one of the most challenging problems in the field.
Khademhosseini imagines that this bottom-up approach will eventually mesh with other approaches, such as the more top-down scaffolding approach. “The level of complex structures we can have now with these techniques is still far away from what natural tissue does,” he said. “No single approach itself is going to be enough to create all the complexity.”
Conflict Disclosure: The authors declare no conflicts of interest.
Funding Sources: The National Institutes of Health, the Coulter Foundation, the Center for Integration of Medicine and Innovative Technology, and the Institute for Soldier Nanotechnology. Yanan Du received funding from the U.S. Army Construction Engineering Research Laboratory, Engineering Research and Development Center.
