Faculteit Dierengeneeskunde

In a series of interviews with researchers of Utrecht University, Utrecht Medical Center or other ULS-partners, we want to give more insight into current developments in the replacement, reduction and refinement of laboratory animal experimentation within these research facilities.

January 2016

Biofabricated liver constructs better predict hepatotoxicity

A picture of the 3D-bioprinter with Associate Professor dr. Spee and dr. Ferreira da Costa, manager of the Utrecht Biofabrication Facility

Drug-induced liver injury (DILI) in humans is the most common adverse drug reaction (ADR), caused by a single dose or the prolonged administration of a combination of two or more drugs1. About 15% of Acute Liver Failures are caused by DILI2. Current in-vitro systems are not accurate enough in predicting DILI. In addition, 30% of human drug-induced hepatotoxicity is not predicted by the available animal models3.  These are the main reasons DILI still occurs in the clinical phases of drug development or even after approval for market delivery (postmarketing), making it a serious health concern.

Solutions from Regenerative Medicine technology

In this context, in vitro biofabrication of 3D-printed liver constructs containing living and functional human hepatocytes for drug screening represent a promising solution and might replace animal testing in the future. By using liver biopsies, human adult liver stem cells can be harvested and expanded in vitro. These cells can be differentiated into more mature hepatocytes to be used for transplantation or for in vitro modeling of (patient-specific) drug treatments (called “personalized medicine”). As the functionality of these hepatocytes derived from adult stem cells is limited, biofabrication of 3D-printed liver constructs offers the opportunity to better mimic liver functioning and architecture. In addition, in 3D-printed liver constructs multiple cell types can be combined. It is anticipated that combinations of hepatocytes derived from stem cells with other cell types will improve their hepatic function. This would be a significant improvement of current in vitro hepatic cell lines, that do not maintain hepatic functions because of cell dedifferentiation4.

Biofabrication process

The biofabrication and optimization of such 3D-printed liver constructs is the field of interest of Assistant Professor Dr. Spee and his liver research group at the Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht University. His research belongs to the expertise area Regenerative Medicine & Stem Cells of Utrecht Life Sciences, an innovation network which unites Utrecht University, other research institutes, government and companies. This network facilitates knowledge transfer with several facility services, for example with the Biofabrication facility at Utrecht University, which brings together different disciplines for the development of 3D-printed tissue constructs. Within this facility, Dr. Spee applies the liver organoid technology of the Hubrecht Institute, provided by Prof. Dr. Clevers and Dr. Vries5, to produce and maintain genetically stable human organoids (adult stem cells) in culture. For this purpose, adult stem cells are generated from liver biopsies from liver donors (provided by Erasmus Medical Centre, Rotterdam). The acquired liver organoids can be expanded almost indefinitely and preserve their genetic integrity over months in culture. The liver organoids are printed in conjunction with multipotent stromal cells from the liver (LMSCs), since this combination of cells improves hepatic functions.

The liver organoids are 3D-bioprinted into stable liver cell constructs. The 3D-Bioprinting technology of Dr. Malda and Dr. Costa is used for this purpose (for more details about 3D-Bioprinting, read more here). After printing, support material in the construct is sacrificed, allowing the formation of a porous construct. The porous liver constructs are cultured into perfusable bioreactors that are custom designed in collaboration with LifeTec Group6 represented by Dr. Kock, a spin-off company of Eindhoven University of Technology. The perfusable bioreactor is a special culture system, in which perfusion allows continuous medium replenishment. After treatment with toxic compounds, damage of the liver cells in the constructs is determined to test the bioreactor model for hepatotoxicity. Preliminary results indicate that toxicity can be measured in the outflow of the bioreactors when constructs were subjected to toxic substances. These results will be followed by a testing panel of hepatotoxic drugs in collaboration with Dr. Kramer of the Institute for Risk Assessment Sciences (IRAS).

Future prospects

The quality of the perfusable bioreactors needs to be improved before the liver constructs can be used for drug screening, toxicology studies or patient-specific drug treatments. These patient specific drug assays will be performed at the Wilhelmina’s Children’s Hospital by Dr. Fuchs, where a wide spectrum of metabolic analyses (including metabolomics) can be applied to patient specific liver constructs to evaluate effect of drugs for a specific patient. Cell viability assays have shown that liver cells in the construct remain viable for up to four days after printing. However, Dr. Spee addresses the need to increase cell viability and functionality of the liver cells, preferably for up to four weeks. A four-day period is too short to test for chronic toxicity, which is relevant in toxicology studies or drug testing for particular drugs. With the combination of 3D-printed liver constructs and the perfusable bioreactors, pharmaceutical companies will have the ability to better predict human DILI or perform drug screening assays in the future without burden for experimental animals or patients.

The stem cell research in Dr. Spee is group is mainly financed by NWO/ZonMW, the biofabrication of Dr. Malda by an ERC-grant, the ongoing DILI-studies are supported by EU-FP7.

1: Leise M.D. et al. (2014), Drug-induced liver injury, Mayo Clin Proc. 89(1): 95-106.
2: Tuschl et al. (2008), Primary hepatocytes as a model to analyze species-specific toxicity and drug metabolism, Expert Opinion on Drug Metabolism & Toxicology 4:7, 855-870. 
3: Russo et al. (2004), Liver transplantation for acute liver failure from drug induced liver injury in the United States, Liver Transpl. 10(8):1018-23.
4: Pfeiffer et al. (2015), Featured article: isolation, characterization, and cultivation of human hepatocytes and non-parenchymal liver cells, Exp. Biol. Met. 240(5): 645-56.
5: Huch et al. (2015), Long-Term Culture of Genome-Stable Bipotent Stem Cells from Adult Human Liver, Cell. 160(1-2): 299–312.
6: More information about the custom designed bioreactors of LifeTec Group can be found here.