The making of a liver

Biofabricated liver constructs to predict hepatotoxicity

In a collaborating project between the Faculty of Veterinary Medicine, the University Medical Centre and the Wilhelmina Children’s Hospital in Utrecht, scientists are developing a new platform for testing of hepatotoxicity. Dr. Riccardo Levato and Dr. Bart Spee are joining efforts and knowledge to biofabricate 3D-printed liver constructs containing living and functional hepatocytes for drug screening, which will represent a promising solution and might replace animal testing in the future.

Drug-induced liver injury in humans is the most common adverse drug reaction. These injuries can lead to acute liver failure, and it is estimated that 15 % of liver transplants required after acute liver failure are caused by drug induced liver injuries. Current in vitro systems for prediction of drug-induced liver injury are not accurate enough, and available animal models are unable to predict 30% of human hepatotoxicity cases. For these reasons, drug-induced liver injury still occurs in clinical phases of drug development or even after delivery to market, constituting a serious health concern.



Can you introduce yourself?

I am a biomedical engineer by background, focusing on biomaterials and on how cells interact with them. I design 3D (bio)printing methods to build cell-instructive 3D structures.

What was the aim of this project?

To develop a miniaturized liver model with organoids derived from liver biopsies (patient- specific) to aid drug toxicity testing.

What did you bring to this project?

I brought the 3D printing expertise, to create the perfusion bioreactor and work on the bioprinting of the organoid laden bioink.

Can you introduce yourself?

I am a molecular biologist working with adult stem cells for advanced in vitro models.

What was the aim of this project?

To create an advanced in vitro model mimicking liver function in vivo.

What did you bring to this project?

My expertise is stem cell biology and hepatology, which I want to combine with biofabrication.

Courtesy of Biofabrication

What is the innovation in the platform you are developing?
We are combining the liver organoid technology with bioprinting to create a system for liver toxicity screening and personalized medicine. Adult liver stem cells can be harvested from liver biopsies and expanded in vitro as liver organoids. However, the functionality of the hepatocytes derived from organoids is limited. Providing a three-dimensional environment with hydrogel biomaterials and microfluidics better mimics the liver architecture allowing the organoids to mature and maintain hepatic functions. In addition, in 3D-printed liver constructs, multiple cell types can be combined which mimics the liver in vivo even more.

What are the challenges you have had to face so far during this project? 

Current platforms for toxicity testing need to satisfy specificity requirements. This means they need to be able to react to toxic stimuli like a liver would do; standard toxicity tests have to respond correctly to a minimum number of recognized compounds (like paracetamol), which may not always be the case with in vitro testing systems.
Specificity is hard to achieve, because even if the gene expression matches the healthy hepatocyte profile, the activity of the cytochromes may not be equal to the in vivo situation. We think that improving the satellite environment, by adding the different cell types present in the functional liver, will bring us closer to replicating this specific activity.   In terms of technology, printing organoids had never been done yet, which meant dealing not only with the feasibility, but also with viability issues: as it is often the case with biofabrication, we had to test a large variety of hydrogels to find a functioning equilibrium between printability of the biomaterial and cell survival.

How are the biofabricated liver constructs realized?

Adult stem cells are isolated from liver biopsies (curtesy of Dr. Luc van der Laan, Erasmus Medical Centre, Rotterdam), and used to grow organoids, which can be expanded almost indefinitely and preserve their genetic integrity over months in culture. The liver organoids are printed in combination with liver derived multipotent stromal cells, as this association has shown to improve hepatic function.
The liver organoids or aggregates are mixed with a gel-like material and bioprinted into stable liver cell 3D constructs. To ensure the printed construct is porous (a necessary requirement to permit nutrient diffusion) the cell-laden gel is printed together with a support, water soluble material. After printing the support material in the construct is dissolved in aqueous media, allowing the formation of a porous construct. These liver constructs are then cultured into perfusable bioreactors that are custom designed in collaboration with LifeTec Group (hyperlink). This 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 measured, to test the bioreactor model for hepatotoxicity. Preliminary results have shown that toxicity could be measured in the outflow of the bioreactors when constructs were subjected to toxic substances.

What are the next steps?

We want to further characterize the specificity of the system by a testing panel of hepatotoxic drugs in collaboration with the Institute of Risk Assessment Sciences and Dr. Nynke Kramer, an expert in the translation of in vitro results to in vivo relevance.
We are also collaborating with Dr. Sabine Fuchs from the Wilhelmina Children Hospital. Dr. Fuchs will conduct drug assays at the WKZ, utilizing a wide spectrum of metabolic analysis that will be applied to constructs of patients with specific metabolic livers disease in order to evaluate the effect of drugs for a specific patient.

This system allows to test specific toxicology responses from a larger number of donors, significantly increasing the chances of detecting possible adverse reactions before drugs reach the market.
By combining the 3D-printed liver constructs and the perfusable bioreactors, pharmaceutical companies will have the ability to better predict human drug induced liver injuries or perform drug screening assays in the future without burden for experimental animals or patients.
Furthermore, one of our collaborators, Dr. Kerstin Schneeberger, has recently received funding (VENI) to scale up the platform and work on whole organ engineering, so that in the future we might be able to use this system to obtain vascularized liver tissue.

This research is funded by NWO Domain Applied and Engineering Sciences (AES), project LIVeCON, received by Dr. Bart Spee with co-applicants Prof. Dr. Jos Malda, Dr. Sabine Fuchs, and Dr. Nynke Kramer.

To find out more about the science behind the biofabricated liver constructs, you can read these publications:

Converging biofabrication and organoid technologies: the next frontier in hepatic and intestinal tissue engineering?

Biotechnology Challenges to In Vitro Maturation of Hepatic Stem Cells

Irina Mancini Written by: