Nanopatterned Human Liver BioChips for Drug Hepatotoxicity Screening

Developing a human liver-on-a-chip device for rapid safety testing of drugs

Drugs and over-the-counter medicines are very safe to take at normal dosage. Pharmaceutical companies spend billions of pounds ensuring newly developed drugs are safe. This process is extremely expensive because there are many steps involved in this assessment, e.g. testing for toxic side effects. Because the liver is the body's main processing factory for drugs, side effects on the liver a major focus of safety testing. Accodringly, before these 'candidate' drugs can proceed to early clinical trials in humans, scientists perform drug testing either in animals or in the laboratory - in culture dishes containing liver cells.

These tests involve seeing if the drug is processed by the liver normally, without any side-effects, or if there is measurable damage to the liver tissue. The liver is the largest internal organ in the body, containing billions of specialized liver cells, called hepatocytes, which are responsible for over 500 metabolic and detoxification functions. Liver damage caused by toxic drug by-products can be very serious and even lead to death.

The best liver cells to use in the lab for drug testing are normal human hepatocytes, isolated from donor human livers. Unfortunately the shortage of human donor organs means very few livers are available for this purpose. As such scientists are developing and exploiting other types of hepatocytes for use in the lab. Drug companies often use rodent hepatocytes for drug toxicity testing. However, due to differences between species, data from any drug toxic effects are not exactly the same as in human liver, potentially missing unsafe side-effects before human drug trials. In addition, human 'immortalized' cell lines are used (hepatocytes derived from sick liver tissue) which are very easy to grow and use for testing in the lab. However, again these cells do not function as healthy hepatocytes do in the liver and therefore, like animal liver data, can fail to predict potential drug-induced liver toxicity. Pharmaceutical companies are therefore constantly searching for other types of hepatocytes for use in drug toxicity testing.

In our project, we will use a novel type of human liver cell line called 'HepaRG'. These cells are special because they form two types of liver cells in lab culture dishes: Hepatocytes plus supporting cells called 'cholangiocytes'. This is very important since as in your own liver, hepatocytes perform best when they are in contact with their neighbours. This actually allows HepaRG cells to perform many biochemical and detoxification functions very similar to the normal human hepatocytes mentioned earlier.

Our main aim in this project is to use HepaRG cells to develop a human 'liver-on-a-chip' device for rapid safety testing of drugs. Scientists know that in the liver, hepatocytes actually respond also to their microscopic environment - even at the nanoscale (one millionth of the thickness of a human hair) - to function better. In our lab, we have shown that cells cultured on special 'nano-patterned' culture slides respond to this environment by changing or even improving their functional properties or rate of growth.

Using different nano-scale patterns we will test many slides to find the pattern which best supports and improves hepatocyte function and appearance. Using the best, or optimal nano-patterned slides, we will then test these using a range of drugs, which are known to be toxic to the liver, to see if they respond in a manner similar to normal human hepatocytes.

Using this information, will then allow us to combine the best nano-patterns into a novel 'BioChip' device, which is compatible with rapid (robotic) drug toxicity testing using special automated cell imaging techniques. Liver BioChips could then be used to test drug safety, reduce drug development costs and specifically reduce the use of animals in drug research.

Conventional drug toxicity models use rodent hepatocytes or immortalized human cell lines, which rapidly lose polarity differentiated phenotype and are not representative of normal liver tissue. Primary human hepatocytes (PHHs) exhibit phenotypic variability and instability in culture, with intermittent supply and high costs. Hepatoxicity is a leading cause of drug attrition, suggesting current preclinical models of toxicity are not universally predictive of drug effects in humans. Therefore a sustainable and scalable human hepatic in vitro cell culture platform would help generate more physiologically-relevant preclinical data for drug hepatoxicity screening. Commercially available bioengineered liver models are based on heterologous hepatic co-cultures, which may be contraindicated for pre-clinical hepatotoxicity testing. We will utilize the human HepaRG bipotential hepatic progenitor cell line as a functional surrogate to PHHs and a unique co-culture system for high-throughput screening (HTS) of hepatotoxicity.
The significance of the proposed work is to develop and validate a prototype HTS-compatible HepaRG-based BioChip by directed nanopattern-guided differentiation. HepaRGs undergo a complete differentiation program, which we will exploit to assess effects of nanopattern-guided differentiation/ cellular reorganization. Thus our research will grant access to general mechanistics of external cues for the improved culture of other hepatic cell types.

We will determine the interplay between nanoengineered substrates and hepatic phenotypic function by assessing:
*Nanotopographical control of HepaRG differentiation on fabricated nanopatterned polymer slides
*Define optimal configuration most predictive of in vivo-like metabolism
*Perform rigorous hepatotoxicity testing interrogated with automated HTS-compatible CellProfiler
*Using the above criteria, integrate the system for HTS using a novel ubiquitous 'BioChip' HTS platform for hepatotoxicity testing

The global market for drug discovery technologies and products is expected to expand from $41.0 billion in 2012 to $79.0 billion in 2017. Indeed, the drug discovery industry is highly dependent on cell-based assays and high-throughput screening (HTS) tools and the latter sector is expected to expand from $11.5 billion in 2012 to $20.0 billion in 2017.

There are some commercially available liver models include from RegeneMed, Hepregen and LiverChip. These systems utilize either heterologous hepatic co-cultures using rodent, primate or primary human hepatosytes combining complex multi-step microfabrication manufacturing processes leading to significantly increase per unit costs.

Besides having direct academic impact, our proposed nanofabrication based high-throughput system will have significant industrial impact. The driving technology underpinning the project is extremely accurate and will provide no batch-to-batch variation between samples. Moreover, it will be based on well established multi-well formats already widely used in industry and pharma. Gadegaard has a number of industrial contacts in the area of plastic cell cultureware made by injection moulding and is experience discussing with industry on translation aspects of the research outputs.

The research to be carried out and the outcomes will also have an interest to the public. With an increasingly aging population we are increasingly relying on the health services provided leading to increased costs. Thus technologies capable of reducing such costs and providing for a faster route from discovery to market for pharma companies will also be of the public's interest. This importance will be communicated to the public using well-established routes of public engagement available at but University of Glasgow and Edinburgh.