Liver Cell Culture System Might Test New HCV Drugs

Liver Cell Culture System Might Test New HCV Drugs
By Michael Smith, North American Correspondent, MedPage Today
Published: January 25, 2010
Reviewed by Dori F. Zaleznik, MD; Associate Clinical Professor of Medicine, Harvard Medical School, Boston and
Dorothy Caputo, MA, RN, BC-ADM, CDE, Nurse Planner

* Note that this study reports a cell culture method that might bypass that roadblock.

Researchers say they can now grow liver cells that maintain their functions long enough to test potential treatments for hepatitis C.

The method uses so-called "micropatterned co-cultures" of primary human hepatocytes and supportive stroma, according to Sangeeta N. Bhatia, MD, PhD, of MIT, and colleagues.

The co-cultures were able to support the entire life cycle of hepatitis C, including infection and replication, Bhatia and colleagues reported online in the Proceedings of the National Academy of Sciences.

Coupled with reporter systems, the co-cultures have "potential as a high-throughput platform for simultaneous assessment of in vitro efficacy and toxicity" of antiviral drugs, the researchers said.

The lack of such a system has been a roadblock to testing potential treatments for the virus, which affects 130 million people around the world, the researchers noted in the journal.

Recently, they added, researchers have been able to propagate the virus in human hepatoma cells, but those cells, among other issues, proliferate abnormally and have disturbed gene expression.

To overcome those obstacles, the researchers turned to primary hepatocytes, which would make a better test system, except that they are notoriously hard to maintain in culture.

To form the co-cultures, Bhatia and colleagues seeded multi-well plates with human hepatocytes, followed several hours later by murine fibroblasts.

"If you just put cells on a surface in an unorganized way, they lose their function very quickly," Bhatia said in a statement. "If you specify which cells sit next to each other, you can extend the lifetime of the cells and help them maintain their function."

In a series of experiments, Bhatia and colleagues found:

* Pseudoparticles bearing the hepatitis C glycoproteins E1 and E2 were able to infect between 1% and 3% of the hepatocytes, but did not infect the fibroblasts.
* A hepatitis C virus modified to express a fluorescent protein persistently replicated over a two-week period.
* Infectious virus was found in the co-culture supernatant from four through 12 days after initial infection.

The researchers also tested some possible therapeutics, including antibodies against viral entry factors and viral protease inhibitors, and were able to show effects on replication of hepatitis C.

They were also able to test two or more drugs simultaneously to show the feasibility of combination drug studies using the system.

Although the system is "an important step forward," Bhatia and colleagues said, the co-cultures have some limitations, including the relatively inefficient uptake of virus.

But they concluded that the co-cultures have the potential to be a "highly valuable system for studies of (hepatitis C) biology."

This study had support from the Greenberg Medical Research Institute, the Ellison Medical Foundation, the Starr Foundation, the Ronald A. Shellow Memorial Fund, the Richard Salomon Family Foundation, and the NIH. The researchers said they had no conflicts.

Primary source: Proceedings of the National Academy of Sciences
Source reference:
Ploss A, et al "Persistent hepatitis C virus infection in microscale primary human hepatocyte cultures" Proc Natl Acad Sci 2010; DOI: 10.1073/pnas.0915130107.