Stephen J. Fey studied cell and molecular biology at King’s College, University of London and obtained his Ph.D. in biophysics in 1980. Interested to understand how living cells respond to external factors, he went to Århus University to study protein expression (now called proteomics). He moved to Odense University in 1996 and was one of the founders of the Centre for Proteome Analysis. When the opportunitly arose in 2006 to spin this institute out of the University into a small biotech company, he took the chance and became the CEO of DrugMode ApS. The company’s products proved to be too early for commercialisation and so DrugMode was closed down in 2011 and he returned to the University of Southern Denmark where he leads the Tissue Culture Engineering Laboratory.
Head of research: Associate professor Stephen John Fey
Assistant professor Krzysztof Wrzesinski
3D Tissue Culture
The ultimate goal of biomedical research is to reduce disease and improve human health. Working towards this goal it therefore makes sense to study how cells work in the human being. It came therefore as a great surprise, that the methods used to cultivate cells in the laboratory (and that had been so successful in the fight against polio in the 1950’s) suddenly were shown to produce cells which poorly reflected what is occurring in our organs. The goal of our research is therefore to develop culture systems that allow cells in culture to mirror what is happening in the body. Such a system will provide a unique window into how healthy cells work; what goes wrong in cancer, diabetes and cardiovascular diseases; and accelerate the development of new safer medicines.
We are collaborating with companies to develop the equipment we need, with other international groups who wish to establish this technology for their own research and with pharmaceutical companies who are interested to use this technology for predicitive toxicology.
Current research projects
Our livers function as an active filter, selecting, modifying and destroying compounds that enter our bodies primarily from our digestive system. Hepatocytes (liver cells) contain a battery of specialised enzymes to do this (called cytochrome P450s). Unfortunately, when hepatocytes are isolated from the body, many of these enzymes become inactive within hours. This means that it is very difficult to test novel drugs in the laboratory and this explains why the pharmaceutical industry has a >90% failure rate for novel drugs when they are tested in clinical trials on humans. We have shown that when hepatocytes are cultivated in the 3D system that we have developed, they provide an assay that is more predicitive of the drug’s toxicity than the assays currently considered to be the best available (“gold standard”). We are currently characterising the performance of the hepatocyte spheroids produced in the 3D culture system in more detail (characterising protein expression, urea and cholesterol production, and cytochrome P450 expression) and benchmarking this performance against the human liver.
Proteomic characterisation of the differences between cells grown in 2D and 3D culture systems
Cellular metabolism can be considered to have two extremes: one is characterized by exponential growth (as seen for example in wound repair) and the other by a dynamic equilibrium (in mature tissue).
We have analyzed the proteome and cellular architecture at these two extremes (exponential growth as characterized by cell growth in 2D culture and dynamic equilibrium exhibited by cells in 3D culture) and found that they are dramatically different. Structurally, actin organization is changed, microtubules are increased and keratins 8 and 18 decreased. Metabolically, glycolysis, fatty acid metabolism and the pentose phosphate shunt are increased while TCA cycle and oxidative phosphorylation is unchanged. Enzymes involved in cholesterol and urea synthesis are increased consistent with the attainment of cholesterol and urea production rates seen in vivo. DNA repair enzymes are increased even though cells are predominantly in Go. Transport around the cell – along the microtubules, through the nuclear pore and in various types of vesicles has been prioritized. There are numerous coherent changes in transcription, splicing, translation, protein folding and degradation. The amount of individual proteins within complexes is shown to be highly coordinated. Typically subunits which initiate a particular function are present in increased amounts compared to other subunits of the same complex.
We have previously demonstrated that cells at dynamic equilibrium can match the physiological performance of cells in tissues in vivo whereas exponentially growing cells cannot. These studies elucidate the molecular basis for these differences in performance and help to explain why growth in 3D is more representative of human tissue.
How does the growth environment influence gene expression, mRNA splicing patterns and protein modification and how does this lead to physiological performance similar to that in vivo?
In classical cell culture techniques the cells are grown attached to the bottom of a plastic flask and are trypsinised at weekly intervals. This predisposes the cell for rapid growth with minimal cell-cell interaction. The 3D microgravity technology that we have developed is designed to achieve the opposite: slow cell growth and maximal cell-cell interaction. To make this possible, we have designed a bioarray matrix that can drive 16 microgravity bioreactors in parallel.
These changes in the ‘external environment’ have been shown to induce a well-established hepatocyte cell line (HepG2/C3A) to recover functionalities characteristic of the organ from which it came (the liver). Some of the effects appear to be induced by a restoration of the epigenetic histone clipping pattern seen in vivo. By using a phospho-proteomics approach, we have started to characterise how these changes in the external environment influence the utilisation of the genetic information. The regulation appear to be exerted via multiple signalling pathways including mTOR; PP1-SRSF (inducing alternate splicing); and GFAT leading to changes in protein O-linked glycosylation. Other pathways like Wnt, p53 and the RAS-REF-MEK do not appear to be involved.
The roles of these pathways is being further probed using redox proteomics in which the drug paracetamol is used to induce redox imbalance, leading to the accumulation of reactive oxygen species and changes in protein S-nitrosylation and S-sulfonylation. These changes will provide a deeper understanding of how our organs work in vivo and what goes wrong in disease. In turn this will open the door to a structured development of new treatments.
Determination of drug toxicity using 3D spheroids constructed from immortalized human hepatocytes.
Fey, S.J. and Wrzesinski, K., Toxicological Sciences, 2012, 127(2), 403-411
After trypsinisation, 3D spheroids of C3A hepatocytes need 18 days to re-establish similar levels of key physiological functions to those seen in the liver.
Wrzesinski, K. and Fey, S.J., Toxicology Research, 2013, 2(2), 123-135.
NB: Manuscript featured on the front cover of the journal.
Human liver spheroids exhibit stable physiological functionality for at least 24 days after recovering from trypsinisation.
Wrzesinski, K., Magnone, C.M., Visby Hansen, L., Ehrhorn Kruse, M., Begauer, T., Bobadilla, M., Gubler, M., Mizrahi, J., Møller Andreasen, C., Zhang, K. Eyed Joensen, K., Andersen, S.M. and Fey, S.J., Toxicology Research, 2013, 2(3), 163-172.
NB: Manuscript featured on the front cover of the journal
A full list of publications by associate professor Stephen Fey can be found here.