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Step Toward a Bioartificial Liver
October 31, 2013   
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Scientists from the Polish Academy of Sciences’ Institute of Biocybernetics and Biomedical Engineering (IBIB PAN) in Warsaw have announced they have built a microreactor for hepatocyte cultures that they hope will prove to be a significant step toward building a “bioartificial” liver—a device that could keep people with acute liver failure alive.

The purpose of a bioartificial liver device is not to permanently replace liver functions, but to serve as a supportive device, either allowing the liver to regenerate properly after acute liver failure or to bridge the individual’s liver functions until a transplant is possible.

Even though liver cells are highly sensitive and difficult to cultivate outside the body, the Warsaw scientists have demonstrated that such cultivation is possible with an appropriately designed microreactor. Their microreactor can potentially be used to build a bioartificial liver device.

In modern laboratories, human and animal cells are usually cultured in standard culture dishes under conditions significantly different from those normally found in living organisms. This makes the proper development of many types of cells difficult. The Warsaw-based Institute of Biocybernetics and Biomedical Engineering has come up with an innovative perfusion microreactor demonstrator for hepatocyte cultures. These liver-derived cells are considered particularly demanding in terms of culture conditions.

“In microreactors, unlike conventional static cell cultures in microplates, it is possible to precisely control the cellular environment and provide cells with the right conditions for growth. However, as hepatocytes have very high requirements, meeting these requirements, even in a microfluidic system, is not easy,” says the institute’s Prof. Dorota Pijanowska, Ph.D.

The microfluidic bioreactors built at the Institute of Biocybernetics and Biomedical Engineering are small plates of transparent polymer that fit in the hand. Each bioreactor is formed by bonding two parts, where one of them is equipped with a system of microchannels and cell culture chambers (created using the so-called replica molding method). Next, cells are introduced into the bioreactor’s culture chambers and the cell culture medium flowing through the system in a continuous manner provides the cells with nutrients and removes metabolites.

Liver cells need a lot of oxygen. Providing them with sufficient quantities of this throughout the culture chamber of the bioreactor may be problematic. The institute’s Barbara Wawro, Ph.D., says, “Due to the relatively low solubility of oxygen in the cell culture medium, in the metabolically active cell cultures, low-flow velocities could result in an oxygen concentration gradient [unequal distribution of oxygen] in the culture chamber.” Close to the medium inlet, cells have enough oxygen, while those located farther away could remain oxygen deficient, according to Wawro.

The problem with providing enough oxygen could potentially be solved by simply increasing the medium flow rate, Wawro says. But a faster flow leads to a greater shear stress, to which hepatocytes are highly sensitive. That is why the researchers had to look for other solutions, she says.

The researchers took advantage of the properties of the polymer from which the microreactor was built, namely polydimethylsiloxane (PDMS), which has high permeability to gas. In the new bioreactor, cells grow on a PDMS membrane with a thickness of 150 micrometers. It is strong enough to allow normal operation of the device, but at the same time so thin that oxygen from the environment can penetrate into the growth chamber.

The height of the chamber—only 100 microns—promotes the proper diffusion and uniform distribution of the oxygen throughout the bioreactor chamber. An additional advantage of PDMS is its transparency. Therefore the microreactor allows continuous microscopic observation of growing cells during culturing.

At the moment, the researchers are working to modify their bioreactor. A team from the £ód¼ University of Technology, responsible for covering the surface of PDMS with various diamond-like carbon layers (DLC) with different textures, is taking part in the project. This type of surface affects the growth of cells and may allow the development of their desired features. In addition, engineers from the AGH University of Science and Technology in Cracow are involved in developing an optoelectronic cell detection sensor.

The Warsaw institute’s Pijanowska says, “There are not too many hepatocytes in a single bioreactor. But our bioreactors are modular and can be combined with each other. In the future, such a complex, after rescaling and other necessary adjustments, could potentially be efficient enough to function as an artificial liver.”

The development of a microreactor for liver cell cultures was one of the aims of the Micro- and Nanosystems in Chemistry and Biomedical Diagnostics (MNS-DIAG) project financed under the European Union’s Innovative Economy Operational Program. The project ended recently and was coordinated by the Warsaw-based Institute of Electron Technology (ITE), which developed silicon master molds for the hepatocyte bioreactor. The project involved 14 research teams from leading Polish scientific institutions, among them technical, medical and agricultural universities as well as R&D institutes and institutes of the Polish Academy of Sciences.
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