It is large, it is heavy, it is meaty. It is, our liver. It is also one of our most vital organs, performing hundreds of functions every day. In drug development, the testing of compounds on hepatocytes – liver cells – is essential to validate their ability to be metabolized and predict whether or not they will result in devastating toxicities. But there is one major problem.
Balanced on top of our digestive system and protected by the rib cage, our liver filters and detoxifies our blood before allowing it to venture into other parts of the body. It also metabolizes drugs. Overall, our liver performs over 500 different functions, working every second of every day as our body’s own detox-machine.
So when our liver fails to do its job, we are in deep trouble. Not surprisingly, biotech and pharma companies make absolutely sure that the drugs we take do not damage it and can be processed by it in the most effective way possible.
In drug development, the liver’s cells – human primary hepatocytes (hphep) – are used as a cell model to observe drug-drug interactions and drug-metabolism activities, as well as to assess the safety of a drug.
Pharmaceutical researchers not only use hphep cells as a model to look at toxicities, they are also a way to screen for new drugs that can be used to treat metabolic liver diseases.
Many diseases require a cell model that helps researchers identify new drugs to treat them. These include treatable genetic diseases, such as alpha-1 antitrypsin (AAT) deficiency, autoimmune diseases, Nonalcoholic steatohepatitis (NASH), hepatitis, fibrosis, cirrhosis and hepatocellular carcinoma. In pharmaceutical research, this process is referred to as disease modeling for drug screening.
However, there is one tiny – or not so tiny – catch: in cell cultures, hepatocytes have a very low survival rate. This makes it difficult to use them for important long-term studies. We have spoken to Dr. Liz Quinn, Associate Director of Stem Cells at the biotech company Takara Bio USA, Inc.
She has spoken to us about the difficulties researchers face when trying to cultivate hepatocytes, why these cells are so important for drug development and what can be done to overcome the stumbling blocks placed in the way of researchers.
Liz, what are the key challenges researchers face when working with human primary hepatocyte cultures?
The biggest problem is that hphep cells don’t survive in standard 2D culture for very long – no longer than 2 or 3 days. Experiments are therefore under a strict time limit. Especially for toxicity research this can be a problem, because once a drug is added, researchers need to observe its effect on the cells for several days.
Although hphep cells can be sourced from patients through a biopsy, this can be logistically challenging. And biopsies don’t always deliver human hepatocytes in sufficient numbers for researchers to perform their experiments. What’s more is that the batch of cells researchers receive are donor-dependent, meaning different donors give different results in experiments.
It is also possible to obtain batches of primary hepatocytes from commercial vendors. However, even though this might be a more convenient and reliable source of cells, each vendor provides their own media to go with the cells.
As a result, no gold standard culture system exists and researchers need to pre-qualify each lot of cells prior to their experiments, which influences the ability to interpret their results.
Takara Bio’s product addresses the short-term culture problem, as well as donor-dependency. Our media has been shown to work across multiple donors equally and across cells from multiple vendors. And this enables researchers to interpret their results from a uniform starting point.
What does albumin secretion have to do with all of this?
Albumin is one of the major readouts for hepatocytes. The hepatocytes sitting in the periportal zone of the liver produce albumin. It is the main protein in blood plasma. Researchers can basically evaluate how well the hepatocytes in their culture are working by determining their metabolic activity, so if and how much albumin they are producing. This analysis tells them whether the hepatocyte cells are viable.
A healthy culture system, therefore, results in viable and functional hepatocytes that secrete albumin. Takara Bio’s culture media does just that. Human hepatocytes growing in our media are able to produce albumin not only for just 2 to 3 days, but up to 4 weeks. This is a great advantage for researchers.
What are the advantages of long-term cultures?
With long-term cultures researchers can do more interesting studies. For example, they can study the chronic effects of their compounds in chronic toxicity studies where they add compounds to their cells and observe how they react over time.
One of the main assays performed by drug metabolism and pharmacokinetics (DMPK) groups are compound clearance assays. Those assays enable researchers to see how quickly cells can metabolise the drug over time. The longer time points give investigators a broader assay window to look at the effects of compounds on the cells.
What makes Takara Bio’s Cellartis Power Primary HEP Medium so unique?
Our medium is the only product currently on the market that can support primary hepatocyte 2D cultures for four weeks instead of 2-3 short days. Hepatocytes are stabilized much longer. Our media results in highly viable, but also metabolically active cells – they have the right morphology and secret enough albumin. Next to albumin, cytochrome P450 is another readout of hepatocytes.
Takara Bio’s culture medium is complete and defined, which means that all necessary components are premixed inside. Researchers can also freeze it. This allows it to be used for multiple experiments. It’s a weekend-free media, which literally means that researchers don’t have to feed it on weekends, but only every other day.
A major advantage is the ability to culture cells in 2D for four weeks and that the cells don’t need an extracellular (ECM) overlay – a matrix containing collagen and hormones, such as growth factors – to stay alive and functional.
The natural function of the liver is extremely hard to maintain in vitro. This means that for normal hepatocyte cultures you need an ECM overlay to support the liver cells and enable their survival. The problem is that many assays aren’t compatible with it. Hepatocytes can live on our medium without the extracellular overlay, as everything they need is already inside the mix.
What kind of companies work with the Cellartis Power Primary HEP Medium?
The type of companies that work with our medium are mainly biotech and pharma companies, including big pharma. Within these companies, there is interest from drug metabolism and pharmacokinetics (DMPK) groups and disease modelling groups looking at animal models and infectious diseases, like hepatitis.
Safety pharmacology groups are looking for alternatives to animal models. There are also many small therapeutic companies focused on regenerative medicine for liver disease. In general, our media can be used by researchers working on infectious diseases, NASH and regenerative medicine.
What are future implications for Takara Bio’s Medium?
Long-term culture of primary hepatocytes can expand the ability for researchers to do chronic, long-term, toxicity studies. With our medium, hepatocytes can stay viable and functional for up to four weeks, allowing the toxicity of drugs to be studied in more detail.
In the future, the media could enable new areas of research, such as hepatitis B and C infections, where long-lived culture systems are required to support all stages of the viral life cycle.
To date, there is no model that can observe the viral binding, infection, cell replication and entire life cycle of the virus properly, because it takes too much time, and until now, cells couldn’t survive long enough. With the Cellartis Power Primary HEP Medium this might succeed.
Also, it is slowly becoming clear that researchers need to know specific ingredients contained in the media, such as growth hormones, when they are studying certain diseases including NASH. Our goal is to create a family of products that are formulated with the proper nutrients, growth factors, components that can support different liver disease areas.
Images via Zolnierek, Giovanni Cancemi, Kateryna Kon, PowerUp, Witsanukorn Oya, Jose Luis Calvo, Jarun Ontakrai, Magic mine, nobeastsofierce/ Shutterstock.com