Artificial liver microbioreactor helps replace animal testing

Research backed by European cosmetics companies looking to find alternatives to testing products on animals has resulted in a device that uses microfluidic technology to simulate how the liver reacts to potentially toxic substances.

The work could eventually help develop artificial structures that combine tissue types from several different organs.

The EU Cosmetics Regulation that came into force in 2013 bans the sale of products containing ingredients tested on animals. One of the most promising alternatives for checking toxicity, which could also be used by the pharmaceutical sector to evaluate potential side-effects of new medicines, involves the use of artificially cultured liver cells.

The EU-funded Hepatic Microfluidic Bioreactor (HeMiBio) project is developing a ‘microbioreactor’ that mimics the structure and function of the human liver, making it possible to assess in real time how effectively the body can metabolise potential toxins.

A key problem until now has been that liver cells rarely survive for longer than a few days in a laboratory environment. This makes it virtually impossible to carry out experiments to determine the long-term effect of toxic substances on a living organism.

In a recent breakthrough, HeMiBio researchers at the Fraunhofer Institute for Cell Therapy and Immunology IZI in Potsdam, Germany, in collaboration with partners at the Hebrew University of Jerusalem, announced that they have been able to keep liver cells alive and under observation for a month.

One of the tasks was to design a suitable reactor vessel containing numerous microfluidic channels, in which all cells are equally supplied with the growth medium so they are evenly distributed and do not form clumps. This created other problems, however, because the wider the distance between the cells, the weaker the signals captured by the sensors.

Faced with the need for a sensing technique capable of dealing with a high concentration of cells, without the risk of introducing interference effects that would falsify test results, the IZI team came up with the idea of using tiny polymer particles containing a luminescent dye.

Exposure to a monochromatic LED light source excites individual electrons in the dye and raises them to a higher energy level. Within a fraction of a second they return to their original energy level, emitting energy in the form of phosphorescent light. Because the time taken for this to happen is directly related to the concentration of oxygen in the surrounding air, it indicates the presence of metabolic activity and can be used to measure the effect of a toxic substance.

By gathering data in real time on the amount of oxygen being taken up by liver cells, the microreactor can create a detailed picture of how metabolic processes are affected by the presence of different ingredients.

Being able to observe this is a major advantage over previous techniques, said Dr Claus Duschl, head of the cellular biotechnology department at the IZI. “Up to now, both in animal testing and in conventional lab tests, measurements have usually only been made at the end of the test,” he explained. “The procedure consists of administering different doses of an active ingredient and subsequently analysing the areas of dead tissue or the dead animal. It is not possible to determine the precise effect of the active ingredient on the cells using this method.”

Researchers have been able to verify various hypotheses about liver activity by selectively replacing specific metabolic products whose synthesis had been blocked by a toxic substance, Duschl added. “As we had surmised, the metabolic process then continued unaffected to the next stage.”

Having proved their device works, the researchers now need to find a way of populating it with combinations of different liver cells to simulate more complex metabolic processes. Eventually, the researchers hope to be able to combine tissue samples from different organs in a single reactor. “But we still have a long way to go,” Duschl admitted.

Other HeMiBio researchers at the Fraunhofer Institute for Reliability and Microintegration IZM in Berlin are already testing a more advanced reactor based on the same microfluidic structures that they have developed in collaboration with colleagues in Belgium. They describe initial results as “extremely promising”.

As well as providing a replacement for testing on animals, the HeMiBio device could offer a source of liver cells for use in the bio-artificial systems used by patients suffering from acute liver failure.

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