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Images of silicon bioreactor containing liver cells
Credit: Ned Jastromb, UCSD

Researchers at the University of California, San Diego have created novel silicon chips with miniature wells similar to those in muffin tins that allow the maintenance of fully functioning liver cells, an important advance for scientists who hope to keep liver cells alive outside of the body.

Their achievement, which could lead to new treatments for liver disease and new methods of testing drug toxicity, will be described at a news conference at the American Chemical Society's 221st national meeting in San Diego.

The development of this dime-sized, porous silicon "liver bioreactor" was the result of a collaboration between chemists in UCSD's Division of Physical Sciences and bioengineers at the university's Jacobs School of Engineering, who suspected that normal liver cells might grow on finely textured surfaces of silicon produced through an electrochemical-etching process.

"This is a great example of how interdisciplinary collaborations can contribute to important advances for human health," says Michael J. Sailor, a professor of chemistry and biochemistry at UCSD.

"We're exploring a new generation of devices in which we can maintain cells by controlling the architecture, temperature and chemical environment, and in which we can use sensors located on the same chip to monitor the health of cells," says Sangeeta N. Bhatia, a physician and an assistant professor of bioengineering at UCSD. Because previous research on porous silicon has been restricted to cancerous cell lines, the porous silicon bioreactor will provide an immediate benefit for Bhatia and her colleagues, who can now study and maintain normal liver cells harvested directly from animals.

It may also help in the development of future artificial liver devices. Today, five companies have artificial livers in clinical trials worldwide. Intended for patients with end-stage liver disease, these external devices house pig liver cells or cancerous cell lines that act as a bridge to keep patients alive until a donor liver is available for transplant. Maintaining live, functioning liver cells has been a challenge in all of these devices, and Bhatia hopes the silicon bioreactor chips will help shed light on new techniques for successfully maintaining liver cells while using them to process blood.

While cancerous liver cells can be easily grown in culture dishes, normal liver cells are much more discriminating. The porous silicon bioreactor design aids in mimicking the conditions found in liver. Individual cells are contained within well-
like structures no wider than a human hair whose surface can be chemically modified to mimic the extracellular matrix distribution found in the liver. The porous nature of the silicon bioreactor can be used to promote the flow of nutrients and chemicals through the cell culture, while filtering larger particles such as bacteria and viruses. "We are trying to develop the conditions necessary to convince a bunch of cells in a dish to behave collectively, the way they do in an organ," says Sailor. Such artificial organs could be used to keep patients suffering from liver failure alive until they are able to receive a transplant, or even until their own liver recovers from injury, avoiding the need for a transplant altogether.

To investigate whether normal liver cells could survive on a porous silicon chip,
Bhatia, who works on various aspects of tissue engineering of the liver, teamed up with Sailor, who is working on novel applications of silicon-chip chemistry. One of Sailor's applications is the production of silicon chips with pores 2 to 1,500 nanometers in diameter. Using hydrogen fluoride and a platinum electrode, researchers in Sailor's laboratory are able to produce tiny cylindrical holes in the chip by electrochemically dissolving parts of the silicon away.

"The materials are made by an electrochemical etching process similar to the reaction that causes your car to rust," says Boyce E. Collins, a postdoctoral student in Sailor's laboratory who headed the research to produce the porous silicon chips. "We have developed methods to control this corrosion reaction on
silicon chips. By changing the electrochemical conditions, we can change the size and shape of the pores."

To determine the types of pores on which normal liver cells might thrive, Collins and Vicki Chin, a graduate student in Bhatia's laboratory, varied the pore sizes on a single chip and observed where the cells aggregated. This experiment allowed them to determine the surfaces that promoted the adhesion of cells to the bioreactor. The 15-micron diameter wells are constructed using a similar masking technique as the one employed to put circuits on computer chips.

The end result is a three-dimensional home for the individual liver cells, allowing the fickle cells to come in contact with the porous silicon on all sides, much as
they would come into contact with the extracellular matrix in liver tissue. Bhatia and Chin have been able to maintain thousands of fully functioning primary liver cells for up to two weeks on the porous silicon surfaces.

Although her ultimate goal is to develop an artificial liver, Bhatia says one of the first applications for the bioreactor chips will most likely be testing the toxicity of experimental therapeutic drugs. One of the liver's main responsibilities is to break drugs into pieces that can be activated to perform their function or inactivated and then eliminated from the body. In general, the metabolism of a drug by the liver will dictate its clinical value. Bhatia says that by introducing various drugs onto the bioreactor, she can test how the substances are detoxified by the liver cells without the need for whole body animal experiments. She can also test the potential for drug-drug interaction by combining two or more drugs on the bioreactor chip.

Techniques developed in Sailor's lab to detect biomolecules can be incorporated on the same chip on which cells are growing to detect whether or not the cells are processing the drugs. Using a small laser, the scientists measure slight changes in the rainbow patterns of porous silicon thin films that contain receptors specific to the drug metabolites. Collins says that because so much is known about the chemistry of silicon surfaces from the electronics industry, receptors can be chemically attached to the silicon to sense a variety of drug metabolites. "These silicon interferometers can detect very, very small changes in color," says Sailor. "If you take a laser that's at the right frequency that matches the properties of that layer, you can measure very small amounts of chemicals as they enter or leave the film."

"Using Professor Sailor's non-destructive light sensing techniques, we hope to get a continuous readout in real time without destroying the cells," says Bhatia. "This is an important feature for drug discovery and toxicity work. It is also important for development of artificial liver devices because right now we have no way of determining whether the liver cells inside the devices are alive and functioning."

Previously, the only way to see how cells were doing were to take snapshots in time, either by destroying the cells or by collecting a sample of the solution and testing it using an antibody, a process that could take up to a day.

The researchers were supported by the David and Lucile Packard Foundation, the National Science Foundation, and the La Jolla Interfaces in Science Fellowship of the Burroughs Wellcome Fund.