Tuesday, 22 October 2019 09:47

News from the EUSAAT Congress in Linz Featured

With around 270 participants from 28 countries, the Congress of the European Society for Alternatives to Animal Testing (EUSAAT) is the largest 3R congress in Europe. It is located in Linz at the Danube, Austria. The congress traditionally takes place in the years between the World Congresses.

Inner-city of Linz.
Photo: Pixabay


With 146 lectures and 77 posters, experienced but also young scientists informed the participants about current developments in the field of 3R (replacement, reduction, and refinement of animal experiments).  Congress participation was actively supported with 20 Travel Awards for Young Scientists.

Well-proven: the congress location at the Johannes Kepler University in Linz.
Photo: Christiane Hohensee


In her opening speech, Vice-president Dr. Dagmar Jírová from the National Institute of Public Health in Prague emphasized the importance of the congress by providing access to current research results, especially for Eastern European countries.



Dr. Dagmar Jírová in her opening speech.
Photo: Christiane Hohensee


In the following, only a few research results and developments can be presented here. For a comprehensive overview, please see the ALTEX Abstract Volume.


Induced pluripotent stem cells: the substance the tests are made of

Prof. Dr. Dr. hc. Jürgen Hescheler gave an overview of stem cells: While research with embryonic stem cells represents an ethical dilemma and is strongly regulated, international scientists turned to induced pluripotent stem cells already some time ago. Today, these cells can already be obtained non-invasively from skin or non-invasive from urine samples.
Therefore, problems with animal cells and the resulting species differences are eliminated. Various disease models can be generated based on the genetic make-up of donor cells. They can also be easily used in automated systems (high throughput) for testing purposes. The European Bank for induced pluripotent Stem Cells (EBiSC) is a quality project and the world's largest collection of induced pluripotent stem cells. These cells are freely available to researchers.

Strong research on neurological issues

A Dutch team of scientists from the University of Leiden has presented its research results using iPSC: The scientists have developed a 3D model of the central nervous system for high-throughput screening of substances to detect neurotoxicity. For the investigations, human nerve and glial cells were developed from iPSC and embedded in an extracellular matrix. The neurons and glial cells were able to connect with each other from 96 individual chips. The model can be used to test drugs, endocrine-disrupting chemicals or pesticides as well as many nutrition-related substances such as preservatives, for their effects on cell function. With their model, the scientists were also able to show that certain chemicals in combination with other substances can have a synergistic, disturbing effect on the development of nerve cells and the formation of synapses.

An Italian research team from Genova used multi-electrode arrays in combination with immunohistochemical investigations to identify, assess and evaluate synapse formation, the release of neurotransmitters and their corresponding receptors on the nerve cells. They have developed a device with which they can collect neurotransmitters in the perfusate. With their in vitro model, they were able to investigate substances for their influence on network activity and release of glutamate of the nerve cells. However, the tests have so far only been carried out with rodent nerve cell tissue. The next step will be to establish the model with human-induced pluripotent stem cells.

Using micro- and millifluidic in vitro models, scientists from the Institute of Technology, Vienna, in cooperation with the Vienna University of Technology, have discovered that the blood-brain barrier (BBB) can be well examined under shear stress, leading to an in vivo similar phenotype. For their research, they tested three models for BBB modeling and found differences inapplicability. For their BBB model, the researchers used the immortalized human brain capillary endothelial cell line hCMEC/D3 and brain-capillary endothelial cells (hiPSBCEC) from human induced pluripotent stem cells.

The young scientist Marco Campisi from the Department of Mechanical & Aerospace Engineering, Politecnico di Torino, Italy, was able to show that nanoparticles such as polystyrenes and polyurethane can cross the blood-brain barrier through receptor-mediated endocytosis. After a while, these particles migrate into the extracellular space. For his investigation, he developed a blood-brain barrier model consisting of a microfluidic microvascular model of iPSC-derived endothelial cells, brain pericytes, and astrocytes embedded in a 3D fibrin gel matrix.

Non-invasive extraction of important renal progenitor cells

Prof. Dr. James Adjaye of the Heinrich-Heine-University Düsseldorf, Germany, has developed a method to extract valuable renal progenitor cells from urine samples. These are rare: only three of these cells can be found in one sample, making a specified procedure essential. Renal progenitor cells have enormous development potential. They can be developed in all possible cells of the complex kidney (podocytes, endothelial cells of the kidney, cells of the distal and proximal tubules, Henle's loop, connecting segments) but also generally in cells of the mesoderm, from which in turn adipocytes, osteoblasts or chondrocytes can be produced.

The cells are genetically very similar to the cells that are available in vivo via biopsies. The kidney precursor cells obtained by the new, non-invasive method are human or patient-specific when they are obtained from the test subject. They can be multiplied indefinitely - a very good source for kidney research. Prof. Adjaye has founded a start-up company called Uricell for the production of and research with these cells.

Innovations in the field of organ-on-a-chip technology

The use of human cells in an extracellular matrix material that reproduces organotypical functional units of the human organism is a major advantage of organ-on-chips over animal experiments. The integration of such organoids into fluidic cell culture chambers enables the mimicking of fluidic in vivo organ-to-organ compounds providing an approach for high-throughput screening.

The Italian start-up company React4Life cultivated 3D ovarian tumor models in a fluidic multi-in vitro organ (MIVO®) device, which simulates the capillary blood flow in the cancer environment that feeds the tumor. The MIVO platform enables reliable drug efficacy testing as an alternative to preclinical animal models as well as analyses of tumor cell invasion and metastasis.  

The research group of Prof. Ute Scheper from the Karlsruhe Institute of Technology has represented a similar approach: multimodal microfluidic platforms named vasQchips for automated and efficient handling. The center of these chips is a curved, porous microchannel coated with endothelium that is connected to the microfluidic flow. The pores enable the supply of nutrients and gases as well as the exchange of growth factors or immune cells with the surrounding compartment. In this chip, different organ models are established and evaluated, including liver, blood-brain barrier, skin or a tumor environment, usable for different applications. The vasQchips enable automated exchange of the controlled culture medium as well as the integration of sensors (e.g. O2, pH). With the dimensions of a multi-well plate, the platform can be easily combined with standard devices such as microscopes or fluorescence plate readers.

Improved liver cell model for efficacy and toxicity testing

Cytotoxicity during drug metabolism in the liver is a serious risk. The assessment before the start of experiments in humans is largely based on in vitro liver cell cultures as a model for in vivo metabolism and animal experiments. However, they often do not accurately represent the in vivo situation and further animal testing is required. In order to improve the relevance of these liver cell models, Prof. Sakai Yasuyuki from the University of Tokyo implemented zonal oxygen conditions. This provides a more accurate in vitro model for preclinical trials that more accurately mimics the in vivo situation and should allow the reduction of animal testing.

New models for components of the immune system

The disadvantages of animal models, in particular for the human immune system, have led to doubt about the predictability of immunomodulation and immunogenicity by the pharmaceutical industry. New "humanoid" models are required for safe pharmaceutical treatments. Dr. Christoph Giese from ProBioGen, Berlin, a manufacturer of biopharmaceuticals, presented the Human Artificial Lymph Node Model (HuALN). It is a microphysiological system that mimics immunity in a continuously perfused 3D culture system, suitable for long-term and repeated dose treatments (e.g. 28 days). It is used to induce or modulate cellular and humoral immune responses as well as to test immune modulation in order to evaluate unwanted immunogenicity responses. It can also be used to evaluate the efficacy of vaccines, adjuvants, and formulations.

Dr. Han-Jin Parki and his team from the Korea Institute of Toxicology, South Korea, succeeded in producing efficient functional and liver-resident macrophages derived from human induced pluripotent stem cells (hiPSCs). They can be used for pathophysiological and toxicological studies. Macrophages play an important role in immune responses and are involved in various pathological conditions. Therefore, the development of tissue-resident macrophages from hiPSCs is of particular interest.

Promising project for basic Alzheimer's research

Prof. Nilima Prakash from the University of Hamm-Lippstadt presented her new research project which has the potential to abandon mouse models in the future. It is assumed that Alzheimer's disease has its causes in neurological development, which requires precise knowledge of the differentiation of dopaminergic neurons during development.
Differentiated cells can be used for the analysis of patient-specific disease phenotypes and for disease modeling to test drugs. The project aims to understand the embryonic genesis of neurons and their survival in adults. It uses genetic mouse models, chicken models, as well as hiPSCs from patients. On the basis of known species differences in gene expression of neuron markers and their function in the developmental context, it is critically evaluated which model is suitable for a better understanding of neuron development in connection with human diseases.

Progress towards animal-free vaccine testing

An important area where animal testing is mandatory is the testing of vaccines to ensure that each batch of vaccine produced corresponds to a batch that has already been proven safe and effective. This is usually done in lethal challenge assays in mice. VAC2VAC, a collaborative research project, which aims to develop and validate animal-free testing approaches, has already been able to develop mass spectrometry assays resp. B cell assays and protein analyses for leptospiral, DTaP (diphtheria, tetanus, whooping cough) and veterinary tetanus toxoid vaccines. Amongst others, progress has been made on immunoassays for veterinary rabies vaccines and tick-borne encephalitis (TBEV) vaccines. To replace the rabbit pyrogen test for the TBEV vaccine, the in 2010 already accepted monocyte activation test (MAT) was adapted and validated. After pre-validation of selected methods by multi-center studies, VAC2VAC aims at regulatory acceptance.

Dr. Dieter Pullirsch from the Austrian Agency for Health and Food Safety (AGES), reported about the decision of the Austrian Official Control Laboratory for Medicinal Products to work on alternative efficacy tests for TBE vaccines. He presented the first results of immunoassays based on specific monoclonal antibodies and suggested a way to implement the tests in the European Pharmacopoeia.

Immunocompetent skin disease models

Young scientists are also working on interesting models. At the University of Würzburg, for example, an immunocompetent skin model based on human cells is being developed in cooperation with the Fraunhofer Institute for Silicate Research, Germany. The aim is to investigate therapeutic options against graft-versus-host disease caused by stem cell transplantation. Graft-versus-host-disease is a cell-toxic reaction of implanted immune cells after transfusions or bone marrow transplants against the host organism. In immunocompromised patients, severe organ damage, as well as skin lesions, can occur. These skin changes are to be investigated and treated with the model.

Model of rheumatoid arthritis

To investigate the mechanisms by which rheumatoid arthritis develops, young scientists from the Charité University Medicine Berlin are working on the development of an in vitro model. First, a healthy joint of bone and cartilage material derived from mesenchymal stem cells was developed. This material is embedded in a synovial fluid consisting of hyaluronic acid with surrounding polycarbonate membranes. In order to produce an inflammation model, immune cells - in this case, neutrophil granulocytes - migrated through the membrane. Then the young researchers measured an increase in inflammation markers in the "joint" as well as markers for tissue degradation processes. In the following step, the researchers want to integrate further immune cell types to test antirheumatic drugs. Another development is aimed to develop an osteoporosis model.

Validation of a test for hormone efficacy

Another interesting congress information was the recognition process for the AR-Calux assay: an androgen receptor test can be used in the future to detect hormone-active substances in vitro. Hormonally active substances can be found naturally in the environment or released by humans from chemicals. They can lead to reproductive impairments, developmental disorders or tumor development. The substances are able to bind to receptors for estrogens, androgens or thyroid hormones. Several Androgen Receptor Transactivation Assays have already been developed to investigate the endocrine activity, of which AR-CALUX, in particular, has proven to be reliable and reproducible. This in vitro test uses the bone cancer cell line U2OS, into which labeled androgen receptors have been incorporated. The test has already been validated by the European validation authority EURL ECVAM and three independent laboratories and recommended for inclusion in a test guideline. A corresponding guideline (No. 458) is now in preparation and will be available by the end of 2020.

News from the implementation of the EU Animal Experiments Directive 63/2010/EU

Dr. Susanna Louhimies from the European Commission reported about the current status of developments in the implementation of Directive 63/2010/EU. The Directive has three objectives: to harmonize conditions of competition for stakeholders in order to improve the competitiveness and innovation of EU research; to ensure high animal welfare standards and implementation of the 3Rs, and to improve transparency to the public on the use of live animals for scientific purposes. The ultimate goal of replacing live animals as described in Recital 10 of the EU Laboratory Animal Directive, according to Dr. Louhimies, is a rather "should" but not a "must" provision.

The European Commission has published two new posters: one on genetically modified animals and one on the stress assessment of animal experiments. These are tips to support the animal welfare bodies of animal testing facilities. Especially the assessment of the degree of stress is a difficult process.

The launch of the publication of non-technical project summaries at a pan-European level will take place on January 2021. There will be a searchable EU database available here. In November of this year, the statistics report with the numbers of laboratory animals will be published. The first of the first EU reports on the implementation of the Directive will also be published in November this year.

The European Parliament has provided additional funding for a pilot project. They will promote the introduction of existing alternatives, facilitate the development and validation of new alternatives, support the exchange of information and knowledge, and provide tools for education and training to facilitate the application of the 3Rs. The pilot project envisages the implementation of six interactive eLearning training modules. EURL ECVAM will coordinate a joint research center to develop 3R resources for high schools, universities and young researchers. In addition, the European Education and Training Platform for Laboratory Animal Science (ETPLAS) will be funded to develop guidelines for evaluation criteria, actual evaluation criteria, and tools for competency assessment.

Refining, reducing and replacing animal experiments during training

In courses on laboratory animal science, training is given on living mice, which are often exposed to increased stress and suffering because of the inexperience of the participants. As part of the 3R refinement principle, simulators for rats and mice have been developed to serve as initial training devices for various techniques before they are performed on live animals. However, current devices do not ensure the adequate acquisition of practical skills and most courses do not use them.

In the research project "SimulRATor" by the Free University of Berlin, all six available rat and mouse simulators are being evaluated in order to define the technical and structural specifications required for the development of a new, anatomically and haptically realistic and cost-effective 3D-printed rat simulator. In parallel, the basic framework and structure of the simulator will be virtually designed and test prints will be made using both hard and soft materials.

Nick Jukes, coordinator of InterNiche (International Network for Humane Education) presented new animal-free innovations for the training of veterinarians, including a dog model for surgical training by the biotech company SynDaver. It offers high accuracy for repeatable surgical procedures and adaptable pathologies that are integrated into the entire vascular system. The Clinical Skills Learning Center in Illinois, USA, has models and simulators to teach a wide range of clinical skills and reduce animal consumption. The equipment is among the most comprehensive in the world. The University of Virginia Tech in Blacksburg, USA, developed an open-source virtual reality program for dog anatomy. Colorado University also offers virtual animal anatomy programs.

Forced application of the 3Rs principle

Kathrin Herrmann from the Center for Alternatives to Animal Testing (CAAT) at Johns Hopkins University in Baltimore, USA, reported on the teaching of animal-free approaches in basic and applied biomedical research. Unfortunately, the Directive 2010/63/EU, which aims to replace all procedures on live animals, does not contain an action plan to achieve this important goal.

The scientific and ethical deficiencies of animal models would not yet be taught to future scientists, and there is evidence that they are not appreciated or simply ignored. Therefore, a comprehensive course has been developed by CAAT. Eight modules cover the main shortcomings of animal models, the full application of the 3Rs principles, the proper conduct of literature research and the planning, conduct, analysis and reporting of research studies. The course also teaches how to critically assess the validity of animal and non-animal models in order to select the best resources for specific research interests. The course will be available online next year.

Kathrin Herrmann also spoke in her presentation "Refinement on the road to Replacement: Are we doing what we can?" about the fact that many scientists who use animals consider refinement to be more urgent and achievable than reduction and replacement. The results of her publication show that much more can and must be done to really apply the 3R framework, in which animal replacement is a clear focus. Recommendations for action are given to accelerate the use of animal-free, human-relevant approaches.

Award for a researcher of FKS-free media

Dr. Jan van der Valk, head of the 3Rs Center Utrecht Life Sciences at the Medical Faculty of the University of Utrecht, was awarded with this year's Björn Ekwall Memorial Award as recognition for his consistent and dedicated work in educating the scientific community on the concerns about the use of fetal calf serum in cell cultures, both from an ethical point of view and because of the lack of reproducibility of results. In this context, he was also honored for his efforts in establishing a database on fks-free media. The prize is endowed with 30,000 SEK.