3R News

3RCC Open Call 2019

The 3RCC thanks all applicants for their submissions!
The center received 96 applications for its 2019 call from about 20 different institutions. The average amount requested was about CHF300,000 and the average duration 2.6 years per project. More than half of the projects are related to replacement.

3RCC to fund six projects worth CHF 1.2 million to advance the replacement, reduction and refinement of animal experiments

The Swiss 3R Competence Centre will fund six projects that aim to replace, reduce and refine animal experiments at Swiss research institutions. The projects include novel approaches to improve cell cultures and organoids that replace animals used in experiments as well as new strategies for surgery training and breeding with the aim to improve animal welfare and reduce the number of living animals necessary. The goal of the application of the 3Rs principle is to replace, reduce and refine, i.e. improve animal welfare, while at the same time generating results that can be more
reliable and reproducible and more relevant to humans.

The 3RCC will support two projects of UZH researchers. Philippe Bugnon (LTK) aims to develop a freely available software tool, which will allow researchers to optimise their breeding strategies. This is especially important when multiple traits are combined, and when the Mendelian laws lead to birth of surplus animals.

Finally, the centre will finance a project in refinement, where researchers explore ways to improve animal care, welfare, and scientific quality. Based on a systematic review, Petra Seebeck (ZIRP, UZH) and Stephan Zeiter from the AO Institute Davos plan to develop guidelines for minimum standards for surgery on rodents. The goal is to make sure surgery is fast, minimally invasive as well as with optimal care as to minimise animal suffering, improve recovery and contribute to the quality of scientific results obtained.

More information

From the Department of Animal Welfare: Dummies instead of Animals? Simulator training for animal experimenters

MausSimulator MausSimulator
Photos: Mouse Simulator (Mimicky Mouse Training Simulator, VetTech) for practicing handling and blood sampling techniques (M. Humpenöder).

In the education of veterinary students more and more simulators are used. While these "dummies" may not completely replace training with live animals, they have the potential to reduce the use of live animals for exercise and to minimize the burden on the animals used by better preparing the students.

People who carry out and / or plan animal experiments also learn the handling of various experimental animal species in Switzerland-wide prescribed courses. This training guarantees a professional and welfare-friendly handling of laboratory animals and includes the exercise on living animals. Simulators can also be used for this training, but the available simulators do not yet offer all the necessary functions.

In the Bf3R-funded cooperation project "SimulRATor", the Institute of Veterinary Anatomy, the Institute for Animal Welfare, Animal Behavior and Experimental Animal Science as well as the Institute for Veterinary Epidemiology and Biometry (Veterinary Medicine, Freie Universität Berlin) are developing a new and cost-effective simulator using 3D print (more information).

In order to adapt the simulator optimally on the course use and aim, currently available rat and mouse simulators are examined for their practical suitability. As part of the close collaboration between the Department of Animal Welfare, UZH and the FU Berlin, in January 2019 tutors and participants of the LTK Module 1 course had the opportunity to test available simulators and to give feedback on their functionality.

We will inform you about published results of the project in the future.

Recent UZH 3Rs research: The CAM-Tumor Assay

CAM

The chorioallantoic membrane (CAM) ex ovo assay of the chicken embryo is an animal-test-free tool, which can be employed, for example, as drug screening platform.  As long as the assay is terminated prior to embryonic day 14 (d14) of the chicken embryo, which we consistently do, the CAM remains free of pain-perceiving nerve fibers, which is the basis for Swiss legislation to not consider it an animal test.

The CAM fuses during avian development from the mesodermal layers of the allantois and chorion and functions as embryonic lung to carry out the gas exchange of the developing organism within the egg. Since this membrane expands very rapidly, it is highly vascularized. As the early chick embryo is naturally immunodeficient, the assay allows explantation and growth of cells from different species without problems of graft rejection. Within the 6-7 day time window (time between cell explantation and final measurement at d14) the assay provides a valuable tool for the rapid screening of the efficiency and safety of drugs that, for example, target angiogenesis, tumor growth and cell dissemination in this setting.1–4. In our project “Co-targeting oxic and hypoxic compartments in canine tumors: CAM models” we are focusing on advancing and evaluating the CAM assay as a valuable tool for the pre-clinical anti-cancer (anti-angiogenic) drug screening, which eventually might contribute to substantially reduce the number of mice needed in pre-clinical cancer research. We are using the CAM assay as ex ovo approach (Fig. 1). For this purpose fertilized chicken eggs are cracked open in a plastic bowl on developmental day 3 (d3). Already at this stage the CAM is visible. At d9, tumor cells are explanted onto the CAM and treated the next day (d10) either by intravenous injection or topical application of different anti-cancer substances used. The experiment will be terminated at d14 to comply with the Swiss legislation. We first aimed to measure if tumor growth is affected by our intervention protocols. This was assessed either by imaging or qPCR. To image the anti-angiogenic efficacy of used drugs and combinations thereof, pictures were taken at d10, d12 and d14 and the tumor area measured. If tumors do not grow in a nice spherical way, which can easily be quantified as proxy for tumor growth, a qPCR approach was performed. To do so, primers directed against the hypervariable D-loop region of mitochondrial DNA against canine (our tumor cell source) and chicken (host organism) DNA were used. With these primers we were able to differentiate DNA between the two species and, that way, could quantify the increasing abundance of canine cells (tumor growth) with normalization to the chicken background. To gain further insights how drugs and drug-combinations we used affect tumor vasculature, we measure real-time perfusion into the tumor explants by means of Laser-Speckle contrast imaging. In principle the tumor and the surrounding tissue is irradiated by low power monochromatic laser light (as the tumor explants grow on the surface of the CAM, they are directly accessible by optical methods), which is scattered by moving red blood cells. The change of laser frequency is analyzed and converted to a color-coded image of the microvascular blood flux (Fig. 2).  To assess our treatments on cell dissemination and tissue hypoxia we follow a rather simplified way, since the assay will only allow us to quantify these parameters within a few day-spanning time window (see above for rationale).  For the measurement of cell spread, we routinely extract DNA from four different zones going outward from the tumor and perform qPCR with the specific primers mentioned above for each zone. That way we can quantify, and normalize, the relative abundance of canine (tumor) cell DNA as a measure of distance from the explant after a very short period of time following the intervention. Regarding the degree of tissue oxygenation, the assay allows us to quantify tumor tissue hxpoxia through the i.v. injection of Pimonidazole (HypoxyprobeTM) and subsequent visualization of hypoxia area and/or intensity either by fluorescent or IHC staining. Lastly, we also succeeded in measuring changes in the vasculature as a response to treatment, by injecting FITC-Dextran complexes into the vessels of the embryo, to obtain information on the vascular length or/and active angiogenesis triggered by the tumor explant. 5,6 We will publish our findings on all these animal-test free assessments of a tumor´s response to therapy in the near future.

In conclusion the CAM-tumor assay is advantageous for a number of reasons. The CAM   is immunotolerant, therefore cells from different species can be assessed as xenografts without being rejected. The assay can be performed in a high-throughput manner to test new anti-angiogenic drugs and combinations, in a short time with regard to their efficacy and safety profiles and allows tumor growth/perfusion to be directly assessed by optical methods. The assay exploits both in vitro- and in vivo-based data acquisition. Lastly, it is a cheap method, which requires no expert knowledge other than good dexterity for egg preparation and drug injection. We, thus, anticipate CAM-tumor assays to be increasingly used to select the most optimal drug protocols prior to mice to help reduce the number of rodents needed in pre-clinical cancer research.

If you are interested or have any questions, please don’t hesitate to contact PD Dr. Thomas Gorr, tgorr@access.uzh.ch

1.           Ribatti, D. The chick embryo chorioallantoicmembrane (CAM). Amultifaceted experimental model. Mech. Dev. (2016). doi:10.1016/j.yexcr.2014.06.010

2.           Ribatti, D. The chick embryo chorioallantoic membrane as a model for tumor biology. Exp. Cell Res. 328, 314–324 (2014).

3.           Deryugina, E. I. Chorioallantoic Membrane Microtumor Model to Study the Mechanisms of Tumor Angiogenesis, Vascular Permeability, and Tumor Cell Intravasation. Methods in Molecular Biology 1430, (2016).

4.           Deryugina, E. I. & Quigley, J. P. Chick embryo chorioallantoic membrane model systems to study and visualize human tumor cell metastasis. Histochem. Cell Biol. 130, 1119–1130 (2008).

5.           Ehrbar, M. et al. Enzymatic formation of modular cell-instructive fibrin analogs for tissue engineering. Biomaterials 28, 3856–3866 (2007).

6.           Blacher, S. et al. Quantification of Angiogenesis in the Chicken Chorioallantoic Membrane ( CAM ). Image Anal Stereol 24, 169–180 (2005).

Recently published UZH 3Rs Research: Biofabricating atherosclerotic plaques

Graphic Summary Mallome 2018
Figure. Graphical summary. (a) Spheroid models mimicking atherosclerotic plaques are biofabricated using a tailored hanging-drop procedure. (b) Model plaques are compared to human native plaques using (c) flow cytometry (vi-SNE: machine learning algorithm used for FACS data analysis) and transcriptomic analysis. (d) Model architecture is verified using immunofluorescence analysis. (e) Spheroid plaques are used to answer biological questions relevant to atherosclerosis research.

Biofabricating atherosclerotic plaques: In vitro engineering of a three-dimensional human fibroatheroma model

Atherosclerotic plaques are inflammatory niches accumulating along the walls of large and small calibre vasculature. Cellular and extracellular composition of human microenvironment and to the recruitment of an invading fibrous layer (fibroatheroma). In the project financed by the 3R foundation we developed a bioengineered three-dimensional spheroid model of human fibroatheroma (Figure). We compared cell-populations in our model to those in plaques from human carotid arteries and we observed remarkable similarities. Additionally, we tested the effects of low density lipoproteins (LDL) on the biofabricated constructs and we observed that low-density lipoproteins affect cell viability and contribute to population polarization towards pro-inflammatory phenotypes. With this project, we established a first human bioengineered in vitro model of late atherosclerotic lesion to be used for the investigation of atherosclerosis aetiopathogenesis. With the spheroid plaque model, we aim at overcoming common hurdles faced by in vitro test systems employed for pre-clinical investigations, specifically in the field atherosclerosis research, but not limited to this arena.  Such model could potentially facilitate prediction of the main triggers of the disease, estimate disease risk levels, determine suitable treatments and ascertain the efficacy of potential treatment options. Importantly, our work supports the 3R principle (Reduce, Replace and Refine): this model could potentially reduce and replace many of the in vivo experiments performed nowadays on animal models, facilitating experiments with human cells retaining higher translational potential. Concretely, the following animal models could be replaced: WHHL rabbits, NZW rabbits, apoE-knockout mice, C57BL/6 mice, LDLR-KO (hypercholesteraemic) mice, Apob100/100 mice, LDL-DKO mice, senescent non- human primates and others.

Authors:  Anna Mallonea, Chantal Stengera, Arnold Von Eckardsteinb, Simon P. Hoerstrupa, Benedikt Webera,
a Institute for Regenerative Medicine (IREM), University of Zurich, Zurich, Switzerland, b Institute of Clinical Chemistry, University Hospital Zurich, Zurich, Switzerland
 
Read the publication

Recently published UZH 3Rs Research: Hydrocephalus simulator

Gehlen 2016
Gehlen, M., V. Kurtcuoglu, and M. Schmid Daners. Patient specific hardware-in-the-loop testing of cerebrospinal fluid shunt systems. IEEE Trans Biomed Eng 63:348–358, 2016.

Hydrocephalus simulator for testing of active ventriculoperitoneal shunts

In the project “Hydrocephalus simulator for testing of active ventriculoperitoneal shunts”, funded by the 3R research foundation (www.forschung3r.ch/de/projects/pr_140_14.html) and based on a former project (interfacegroup.ch/project/biothermofluidics-for-cerebrospinal-fluid-diagnostics-and-control), a hardware-in-the-loop (HIL) test bench was developed that can mimic pathophysiologic intracranial and intraperitoneal dynamics as well as posture changes. On this novel HIL test bench ventriculoperitoneal shunts or new control strategies can be tested.2 Hydrocephalus is a defect of the central nervous system where excessive cerebrospinal fluid (CSF) accumulates inside the brain cavity. Hydrocephalus is treated with the placement of a ventriculoperitoneal shunt. Today, more than 30% of all conventional shunts fail within two years.

While a new generation of intelligent shunts promises to be a major step forward, they also introduce increased complexity into the shunt-patient interaction. The HIL test bench allows to reveal the potential of an intelligent shunt and to prove its safety during the approval process. The HIL concept implies that: (1) The patient’s relevant physiology is replicated in real time by an accurate mathematical model;3 (2) the relevant simulated pressures for a ventriculoperitoneal CSF shunt, which are intracranial (ICP) and intraperitoneal pressure (IPP), are applied to the proximal and the distal ends of the catheter, respectively; and (3) the drainage rate of CSF is measured and fed back to the in-silico simulation in real time.2 A video illustrating the functioning of the HIL test bench can be found at http://ieeexplore.ieee.org/ielx7/10/7384657/7160680/tbme-schmiddaners-2457681-mm.zip?tp=&arnumber=7160680.

By reproducing the problem of siphoning and its avoidance with anti-siphon devices, we have proven that this HIL test bench allows for fast, cost effective, and realistic in-vitro testing of active and passive shunts.1 It permits to quantify a shunt’s performance within a realistic yet reproducible testing environment avoiding animal experiments (see www.pdz.ethz.ch/research/biom/smartshunt.html and interfacegroup.ch/project/smartshunt-the-hydrocephalus-project).

Contact: Marianne Schmid Daners1, Vartan Kurcuoglu2,3

1 Product Development Group Zurich, Department of Mechanical and Process Engineering, ETH Zurich, Zurich, Switzerland, 2 The Interface Group, Institute of Physiology, University of Zurich, Zurich, Switzerland, 3 Neuroscience Center Zurich, and the Zurich Center for Integrative Human Physiology, University of Zurich, Zurich, Switzerland

Read more: Publication 1, Publication 2, Publication 3