Interview conducted by April Cashin-Garbutt, BA Hons (Cantab)
Medicyte have recently announced that they are undertaking an EU-project (Re-Liver) with the aim of designing a biomimetic bioartificial liver. Please could you explain to us what one of these is and what it would be used for?
Biomimetic bioartificial livers are liver organoids based on synthetic equivalents of the human extracellular matrix (ECM) which will be seeded with human cells.
By combining novel aspects of material design, cell proliferation and culture technology, organoids will be developed for implantation by minimal invasive injection and providing essential cell function. These artificial biomimetic liver constructs will be reconstituted for in vitro applications and for the treatment of metabolic liver diseases.
The project combines the innovative expertise of the companies Medicyte and The Electrospinning Company with the extraordinary competences of the involved academic institutions University of Manchester and Universita di Pisa. The SME GABO:mi will contribute to the success of Re-Liver ensuring professional project management.
Why is there such a shortage of donor livers and how does this compare to the donation of other organs?
According to UNOS (United Network for Organ Sharing), the number of patients awaiting a liver transplantation has increased more than 15-fold over the last 10 years (now about 16,000 patients ), whereas in the same period, the number of liver transplants increased less than 2-fold only to about 4,000 transplantations. The situation for kidneys is even worse. In the U.S. more than 90,000 patients are listed for a kidney transplant, but only about 16,000 patients receive a kidney each year and about 4,500 patients die while waiting.
Tissue engineering could offer a potential alternative to treat tissue loss or organ failure by implantation of an engineered biological substitute, so called artificial organs or tissues, to restore or re-establish normal functions.
What diseases could potentially be treated by a biomimetic bioartificial liver transplant?
The first target for the organoids are patients with genetic metabolic dysfunctions where cells comprising less than 1-3% of total liver mass are required to increase essential liver function and to achieve significant beneficial outcome. The product could then be expanded finally to treat patients with liver failure who require around 20% of total liver mass or more, which would be provided by injecting multiple bioartificial liver organoids.
Would the biomimetic bioartificial livers last for life or would they need to be replaced at intervals?
This is one of the questions we are going to address in our future (clinical) studies.
How long do you think it will take to develop a biomimetic bioartificial liver?
The three year research project (Re-Liver) should enable our consortium to reconstitute artificial biomimetic liver organoids for in vitro applications and to demonstrate pre-clinical proof of principle in animal studies allowing clinical studies to begin.
Medicyte has also recently been in the news regarding their plans to develop a next generation predictive 3D cell toxicity assay. Please could you tell us a little bit about cell toxicity assays and what they are used for?
One of the main causes of drug failure and removal from the market is liver toxicity. Clearly, more predictive screening models are needed to detect such a serious side-effect.
Cytotoxicity/hepatotoxicity assays should give information on the concentrations at which a drug causes an effect on hepatocytes and whether the effect is affected by metabolism – either detoxified or bioactivated.
The cytotoxicity assays that we conducting are based on a 96-well plate format, which enables us to test the toxicity of a range of concentrations of ten compounds in hepatocytes from four donors (using only one vial of cells for each donor) in a single experiment.
We measure four endpoints to allow us to detect the toxicity of compounds which have different mechanisms of toxicity: morphology; mitochondrial function (MTS metabolism); ATP content and lactate dehydrogenase release.
By screening compounds, we can flag them as being non-toxic, moderately toxic or severely toxic. We can extend these assays to include the effect of drug metabolising enzyme inhibitors to determine how these are involved in the bioactivation/detoxification pathways.
What is the difference between 2D and 3D cell toxicity assays?
The methodology of 2D and 3D cytotoxicity screening is exactly the same! Both can be adapted to liquid handling machines and both can be used to screen multiple compounds.
The main differences are that the cells are plated and penetrate a scaffold in the 3D 96-well plates and a higher cell density is used for the 3D cultures (since they are a multi-layer culture).
How is Medicyte GmbH working with Reinnervate Ltd to produce 3D cell toxicity assays?
We are working together to develop a 96-well plate screening assay which, by combining the alvetex scaffold technology with Medicyte’s upcyte cells, will result in a model that can detect the hepatotoxicity of compounds which was not detected in traditional 2D monolayer primary cultures of rat or human hepatocytes.
We have selected a number of compounds with known in vitro hepatotoxicity and/or have failed on the market due to liver toxicity and screened them in our 2D and 3D screening models. Early data suggest that placing 3D increases the sensitivity of the cells to hepatotoxicity, possibly by increasing the metabolic capacity of the cells.
You are quoted as saying “the combination of both technologies will undoubtedly lead to a more predictive culture model without the limitation of cell supply”. Please could you explain to us exactly how this will occur?
We realise that screening compounds requires a huge source of cells but if these are not relevant or predictive (such as HepG2 cells), then the screening method may select potentially hepatotoxic compounds and deselect promising compounds.
Upcyte hepatocytes can be produced in the quantities needed for screening and have been shown to be more sensitive than HepG2 cells to certain hepatotoxins (for example α-amamitin) and can be used to determine the contribution of CYP3A4 in bioactivation of compounds (e.g. aflatoxin B1).
An important aspect to consider is the inter-individual variation in responses and this can be studied using upcyte hepatocytes from different donors – something that is not possible with cell lines which are from a single donor. Therefore, upcyte hepatocytes are a relevant cell type with drug metabolism characteristics more closely resembling primary human hepatocytes than other screening cell types.
Our studies have shown that upcyte hepatocytes benefit from being placed into 3D culture such that their responses to known CYP inducers are increased when compared to 2D cultures. The 96-well alvetex plate format will allow us to culture the cells in a more in vivo like manner and therefore increase the predictivity of the screens.
Are there any other benefits of working together with Reinnervate on this project?
By generating these important screening data, we can provide support for the use of 3D models for earlier compound testing. It is a misconception that 3D culture is complicated – at least with alvetex scaffolds – and our findings will hopefully encourage more researchers to consider this technology earlier in their screening process.
What medical benefits might this work bring?
Ultimately, a more predictive screening model will result in fewer hepatotoxic drugs moving too far into the drug development process and entering the market.
What are Medicyte’s future goals?
Medicyte’s goal is to be the preferred supplier of different cell types needed for in depth ADMET (Absorption- Distribution – Metabolism – Elimination – Toxicity) investigations, including hepatocytes, gastrointestinal and renal epithelial cells. To obtain optimal results Medicyte offers further supportive cell types, such as upcyte® microvascular endothelial cells.
Upcytes® combine advantages found in cell lines - such as ease of handling and the capability for up-scaling, with the desired features of primary cells, which represent the natural cells of the human body. This bridges a currently existing gap in basic research and industrial applications such as drug discovery and drug development.
Where can readers find more information?
They can visit the websites: www.medicyte.com, www.reliver.eu
About Dr. Joris Braspenning, PhD, Managing Director
Joris is a biochemist (PhD from Leiden University, Netherlands) and has been working in life sciences for more than 15 years.
After working at the Deutsches Krebsforschungszentrum (DKFZ) and postdoctoral studies at the University of Heidelberg, he has been working for more than 7 years in senior positions including Head of Biochemistry and Cell-Biology in the pre-clinical development department at Heidelberg Pharma AG.
His professional experience also includes several years of project management in pre-clinical drug development. Joris is Co-Founder of the Company.