Livers in the Lab

With an unusual background spanning medicine and mechanical engineering, HHMI investigator Sangeeta Bhatia has taken three-dimensional (3-D) culture methods to new, higher-tech levels. Among her broad interests is regenerative medicine; she wants to grow human livers in the lab.

Recreating a cell’s natural environment in the lab means providing four things, says Bhatia: the fibrous matrix or “scaffolding” that supports cells, a community of cells of the same and different types, a dynamic flow of soluble chemical signals, and the interplay of forces, such as shear stress, in the 3-D context.

Over the past decade, Bhatia and her colleagues have moved the field forward, culminating most recently in the creation in her lab of small functioning livers that have been implanted in mice.

Step by step, the team has overcome many hurdles to make hard-to-please liver cells happy in laboratory culture. (In fact, many normal cells are harder to maintain than cancer cells, which are “in the business of being immortal,” Bhatia observes.)

In the early 1990s, Bhatia, working with her advisor Mehmet Toner, borrowed microscale methods used in the computer chip industry to micropattern proteins and cells of different types on two-dimensional (2-D) surfaces. “Cells are about 10 microns wide, and for semiconductor technology, manipulating things on that scale was trivial,” she says.

Another computer chip technology, photolithography, helped her get human liver cells to thrive in colonies on a 2-D surface. Bhatia and her team organized different patterns of liver cells mixed with collagen matrix onto glass plates. After systematically varying the geometry and studying the impact on liver function, they succeeded in sustaining growth of circular colonies of 250 cells, spaced 1 millimeter apart.

Both the relative positions of the cells and the timeframe proved to be key variables. Using a device shaped like two miniature interlocking combs, the researchers were able to position liver cells and supporting “stromal” cells so that they were actually in contact or, alternatively, sending signals across a tiny “moat”—about four cell widths—separating them. The liver cells needed to touch stromal cells for 18 hours before they would express key functions; after that, staying close but not in contact was sufficient.

“Our goal is to put the cells in a device to support a patient with liver failure,” explains Bhatia. “So we needed to know, do you need to implant the supportive cells all along, or just in the beginning?” Since then, she adds, “We have identified about 20 candidate proteins that might be able to replace the ‘closeness’ with a media additive or a tethered factor in the implant.”

These flat cultures have proved valuable for testing drugs against hepatitis C and malaria, among others, and studying drug metabolism. With this system, the Bhatia group along with Charles Rice, a Rockefeller University virologist, was the first to achieve a sustained, productive infection of liver cells in vitro with the hepatitis C virus.

High-Tech Jell-O

To grow a functional liver meant moving to 3-D methods. Bhatia and her colleagues adopted a method similar to one used by HHMI investigator Kristi Anseth, who had been a postdoctoral fellow with MIT biomaterials pioneer Robert Langer. Bhatia and her colleagues made use of these light-activated, cell-containing extracellular matrix gels. “You shine a light on them [from outside the body] and they turn into Jell-O,” explains Bhatia. “The cells are the fruit in the Jell-O.”

After five years of work, the team succeeded in making a “bioactive scaffold” by coculturing liver cells, mouse embryonic fibroblasts, and adhesive peptides in the gel. Bhatia and colleagues also devised a clever way to position cells within the scaffold from outside the body.

“We recognized that when you disperse the fruit in the Jell-O it’s completely unorganized,” she says. “We wanted to organize the cells in three dimensions.” They used electrical fields to move cells into the desired configuration. “You can hold them there and specify their shapes, and then they get frozen in place by the light.”

Ultimately, Bhatia envisions “printing organs” layer by layer in much the way some plastic prototype parts are fabricated. It won’t be enough just to have hepatocytes organized and functioning, she explains. They also need to be able to drain the bile the liver cells excrete and communicate with the bloodstream to detoxify wastes. To achieve this, her team is incorporating other cell types, including progenitor cells that can form the cells that line bile ducts.

Using a variety of high-tech tools, Bhatia has taken a major step by creating “little livers” in the lab. They are about the size of the bottom of a soft drink can. The raw material is her light-sensitive matrix populated with liver cells and endothelial cells. The livers are sculpted with computer-controlled photolithography machines that build up the organ one tiny later at a time—a sort of 3-D printing process.

When Alice Chen in the Bhatia laboratory implants the sculpted livers in mice, the organs do most of the things that livers need to do: connect to the circulation, secrete human proteins into the blood, and metabolize drugs. For now, they live only a few weeks, but new degradable materials are being incorporated to extend their lifetime and potentially even allow them to grow into bigger structures.

Although there is a long road ahead, Bhatia is optimistic that this momentum will continue toward personalized artificial livers that someday could alleviate the shortage of donor organs.

-- Richard Saltus
HHMI Bulletin, August 2010

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