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Zahn-Organoid. Invagination von den Epithelzellen in das Kondensat aus den Zellen der dentalen Pulpa. (Foto: J. Rosowski/TU Berlin)

Funding line "Adding 3R value"

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Funding objective

This kind of funding is aimed at complementing existing research projects at the Charité by an aspect of 3R values – be they sponsored by the DFG (German Research Funding Organisation), the BMBF (Ministry for Education and Science) or the European Commission. It runs on the assumption that many research projects utilize methods that have the inherent potential to drive research in 3R as well. As research projects, which are sponsored by third-party funds, are usually restricted to examine a specific scientific hypothesis(the examination of a certain cellular mechanism, for instance), 3R-related research by rule cannot be sponsored within such a project. Charité 3R wants to close this gap by co-funding projects which have the potential to expand their scope by an aspect of 3R.

Currently funded projects

Replacement, reduction, refinement - Machine learning as a novel tool to tackle mechanisms of atrial fibrillation

In this Project, the researchers aim to develop a machine learning (ML) tool to analyze the Gq pathway related substrate for atrial fibrillation (AF) and predict AF recurrence after invasive and non-invasive treatment. The proposed ML tool will be used to analyze data from patients as well as from wild type and Gq knockout mice (as available from the associated DFG grant), to predict left atrial volume, fibrosis, and the likelihood of atrial fibrillation recurrence. It will also add to the existing knowledge of the Gq pathway and related pharmacological approaches.

The proposed ML tool will replace, reduce, and refine the use of animals in research. By using pre-processed data from electrocardiography, echocardiography, left atrial angiography, hemodynamics, and 3D-electroanatomical mapping, the ML tool will be able to identify novel patterns, and might yield more accurate predictions than traditional animal experiments. Random shuffling, allows to further reduce the need for animal experiments. The ML tool will also be made available to other researchers via the BIH Translation Hub Digital Medicine.

This project might lead to a better understanding of AF, the underlying Gq pathway and ultimately could provide other translational cardiologists with a novel tool for the assessment of AF

Refining analgesia through combined and measurable score systems for inflammation, pain, and inflammatory signalling

Animal models are important and irreplaceable for studying complex multi-organ diseases, however most are performed either without analgesics or without consideration for possible interference with the processes studied. Here, the reserchers propose a pipeline to refine analgesia in inflammatory conditions by evaluating: A. analgesic efficacy B. systemic inflammation C. activity of a key pro-inflammatory pathway, NF-κB, in a mouse model of colitis, as an example of a process studied.

Each score comprises at least two independent and validated methods, thereby providing a robust system for analgesia selection. Analgesic efficacy will be determined by mouse grimace scale (MIG) and pain score. Inflammation will be quantified by well-established histomorphological scoring, and by quantitation of tissue leukocytes. NF-κB activation will be quantified in a reporter mouse model and validated by qPCR. The combined score will identify best-fitted analgesics. This pipeline can be adapted to other animal- and disease models and therefore will have a wideranging impact. Importantly, refinement measures are vital to 3R because they permit the utilization of potentially pain-inducing, but important disease models by alleviating suffering.

The research described will be disseminated via open-access publications, seminars, and public outreach.

Development of full spectrum flow cytometry panels to reduce animal numbers in liver cancer immunotherapy research

Hepatocellular carcinoma (HCC) is among the most common cancers worldwide with increasing mortality and limited treatment options. Since hepatocarcinogenesis is complex, we are dependent on animal models for pre-clinical research aimed at developing novel therapies. We are investigating immunotherapies in mouse HCC models and use flow cytometry to analyze immunological changes as well as therapy responses. However, conventional flow cytometry is limited by the number of parameters that can be analyzed in a single sample. Thus, large animal numbers are usually required to collect enough material to cover all immune cell subsets, also preventing repeated sampling of the same animal. In contrast, full spectrum flow cytometry (FSFC) enables to tremendously increase the number of parameters per sample.

Here, the researches aim to use this new technology to develop and optimize panels that enable analysis of all major leukocyte subsets in one panel. This will reduce the amount of blood per sample and enable repeated testing of the same animal. Therefore, we can significantly reduce the number of experimental animals needed; we can also better mirror the clinical situation for translation (“on-treatment monitoring”). They also plan to develop analogous FSFC panels for human samples to ensure translation of the pre-clinical studies.

Projects with completed funding

3D organoids in head-throat-tumors and in breast cancer

Immune therapy is a therapeutic strategy which is based on the specific modulation of the immune system. It is of great interest in oncology. So-called checkpoint inhibitors, which release certain brakes in the immune system, are considered especially promising. Whereas some of these drugs are already approved for the treatment of certain cancer types, additional (pre-)clinical studies are needed to examine their effectiveness, doses and toxicity. Doing so, the choice of an appropriate model system for the testing of novel immune therapeutic approaches is very important. Lacking alternatives, scientists often resort to mouse models such as patient-derived xenografts. But these models are only partly suited, since they neither reflect the human immune system nor the immune (micro) environment. It should also be noted that for these experiments a large number of test animals is required.

This is why the team around CCCC director Prof. Dr. Ulrich Keilholz plans to establish an alternative derived ex vivo from human tumor tissue: in a first step within the framework of the 3R project , so-called patient-derived 3D organoids for head-throat-tumors as well as for breast cancer are developed. Existing protocols are optimized for this measure. Adding to this, the cancer researchers want to generate immune-competent ex vivo models that have the properties of a functioning immune system. The human models are then employed for the initial testing phase and are supposed to replace much animal testing in the future.

Antiviral tests on the human lung model

Pneumonia is still one of the most prominent causes of death worldwide among children and adults. About a third of all pneumonias contracted outside the hospital are triggered by viruses, above all influenza viruses. The development of new antiviral medication is thus a pressing issue. Dr. Katja Hönzke, Prof. Dr. Andreas Hocke and Prof. Dr. Stefan Hippenstiel are trying to prove in a study that this can be done without animal testing and through the use of so-called ex vivo lung tissue. It should be noted that the tissue samples come from the patients themselves; in this case from co-operations with four local thorax surgical clinics. On these human lung models the scientists test established medication as well as completely novel substances for their effect on the replication of the influenza virus and the antiviral immune reply. The results are then compared with data from in vitro and animal models across the literature. It is the scientists’ goal to show that human lung tissue cultivated ex vivo is generally suitable for the testing of new medication – and can thus replace a part of animal testing.

Artificial intelligence in the development of pain relievers

The study group of Prof. Dr. Christoph Stein has developed new pain relievers (NFEPP and derivatives) with the help of artificial intelligence(AI), which activate opioid receptors only in the acidic environment of peripheral injured body tissue. Healthy tissue in turn does not react to the agent. The central side effects of ordinary opioids such as sickness, fatigue, dependency, addiction and respiratory arrest can thus be prevented. In therapy, opioids are often administered together with anti-inflammatory non-steroid antirheumatics (NSAR). Common representatives of NSAR are Aspirin, Diclofenac or Ibuprofen. The scientists now want to examine whether NSAR influence the effectiveness of the newly developed NFEPP and how its chemical structure may be altered to achieve optimal pain relief when administered in combination with NSAR. Animal testing is usually imperative for such pre-clinical tests. But in this 3R project the change in the acidic environment through NSAR is simulated via computer through AI-based methodology. Doing so, the number of required animal testing can be greatly reduced. Stein and colleagues closely co-operate in the development of the new pain reliever with the Zuse Institute for Applied Mathematics in Berlin (Marcus Weber).


Brain research with genomic single cell analyses

How does brain repair work in humans? How can physicians support these mechanisms when treating patients with neurological conditions?

In order to answer this question, scientists must first understand what brain cells are responsible for the repair – a question, which has been answered with animal testing in the past. To do so, cells first had to be experimentally marked and then trailed. A new method now allows for intelligence on the fate of cells also in retrospect with the use of genomic single cell analyses. In theory, this method makes it possible to even use tissue of the deceased.

Dr. Sarah-Christin Staroßom now wants to utilize the genomic single cell analyses for examinations of the human brain. She is being supported in the development of this alternative method by Dr. Leif Ludwig of the Broad Institute of Boston/USA. In the long run, the scientists hope to gain a better understanding of the repair processes in the brain as well as gaining a better command of developing future therapies. This new method also offers an excellent opportunity to drastically reduce the number of the required animal testing.

Human neuroblastoma 3D model

The neuroblastoma is the second most common solid tumor in infancy, sadly still resulting in too many infancy deaths. Thus, innovative therapy options are necessary to increase the likelihood of survival when faced with this illness. The so-called CAR-T-cell therapy gives rise to new hope. It is a certain kind of immune therapy, where T-cells of cancer patients are genetically re-coded and equipped with a so-called chimeric antigen receptor(CAR).

In this funded 3R project, the team of PD Dr. Annette Künkele will examine the functionality of the CAR-T-cell therapy in a 3D model of the human neuroblastoma. In this model, tumor cells and endothelial cells are printed into a 3D construct together with blood cells. The CAR-T-cells will have to break this barrier in order to reach the tumor. This is a problem that adds to the uncertain therapy success of CAR-T-cells with solid tumors. The scientists will turn off relevant effector genes in the blood vessel wall via CRISPR/Cas 9 technology at a later date to examine their effect on the CAR-T-cell therapy. The scientists are looking to prove in this project that this new 3D model can greatly reduce the number of CAR-constructs that need to be validated in pre-clinical animal testing. The project is a cooperation between Charité scientists and the Berlin startup Cellbricks of the TU Berlin.

Living bio bank from blood progenitor cells

Pre-clinical examinations of animals add to the understanding of pathological developments in the living organism. Many of these examinations could generally be performed on biological material from humans such as cells, tissue or organs, if suitable models were developed and optimized. This is also true for leukemia and further diseases of the blood forming system. In this funded project, Prof. Ulrich Keller builds a living bio bank of human blood progenitor cells through methods of cell culture. Keller and his team use the so-called “HoxB8” technology, which make blood cells immortal, as well as the CAS9 system, with whose aid blood cells may be genetically altered in order to behave like leukemia cells.

The human model allows for the simulation and functional examination of normal occurrences in the development of the blood forming system as well as of pathological processes triggered by genetic alteration. With the living bio bank the scientists want to build the foundation for future research projects of the blood and immune system and to reduce examinations on living organisms – that is, animals. In the long run, this model shall serve cancer researchers to develop better therapies for leukemia, lymphoma and myeloma(cancer of plasma cells). Co-operating partners are the Berlin Institute for Health Research (BIH), the Max-Delbrück-Centre for Molecular Medicine (MDC) and the Technical University Munich as well as the universities of Frankfurt and Würzburg.

Medical imaging of the cardio-vascular system

3D image of the forking aorta of a pig: new imaging data reduce the number of animal testing (Image: Priv.-Doz. Dr. S. Niehues, MHBA)

The improvement of animal models is another way to reduce animal testing. This project strives for a better understanding of the anatomy of the cardio-vascular system in pigs, which plays a large role in cardio-vascular research. Up to now, most knowledge rests on autopsy studies of dead animals. Project director PD Dr. med. Stefan M. Niehues and his team do think that the cardio-vascular system of living pigs (in vivo) differs from the ex-vivo situation (examinations from autopsies) and from the human anatomy as well. Scientists should be knowledgeable about these differences, before using pigs as an animal model for vascular procedures or operations, unless such experiments might prove useless. The scientists also suspect that there are physiological differences in the cardio-vascular system, depending on whether the pig is lying in prone or supine position.

CT examinations of the central cardio-vascular system of pigs shall provide a clearer picture to what degree the in-vivo anatomy of the vessels differs in relation to the positioning of the pigs. The radiologists also use a new technology in the imaging of the vessel system, the so-called “Global Illumination”, which visualizes the arterial as well as the venous system in a compact fashion and thus offers detailed and realistic information on the anatomic environment. The scientists want to use the newly won data for more precise planning of animal testing and to sooner forecast, whether the chosen animal model is suited for the project or not. Many unnecessary tests on animals could be prevented using this new technology.

Microfluid chip for the modeling of acute renal failure (hAKI chip)

About 30 percent of all hospitalized patients develop acute renal failure(ARF). In acute renal failure, a sudden decrease in organ function occurs, which may lead to a total loss of the kidney function. Involved in this scenario are the cells of the kidney tube system, the so-called proximal tubule, immune cells as well as cells of the surrounding blood vessel system. In pre-clinical studies regarding ARF, animal models are most commonly used, because of the complex cell interactions. The rising number of test animals required and the lack of success to translate the findings into the human system call for the necessity of alternative models. Andreas Kurtz and Bella Roßbach of the BCRT work on an animal-free alternative: in this 3R project, the two scientists develop a so-called microfluid chip-based platform(hAKI chip), which closely resembles the human in-vivo situation, meaning that it largely simulates the human organism. In order to do this, human induced pluripotent stem cells(hiPSC) are differentiated into proximal tubule cells as well as endothelial cells and pericytes typical of blood vessels and then fed into a chip capable of perfusion. In the model, the acute renal failure is triggered by stress markers which also occur in humans, such as oxygen shortage or the renal toxic agent Cisplatin. This humanized ARF-model may replace animal testing in the future and help bring new therapeutics more quickly from the laboratory into the clinic.

Multiplex microscopy in immunology

Example of a multiplex histological analysis(MELC) of the human tonsil. 37 parameters are colored on the same tissue section portraying immune cells as well as the surrounding binding tissue. (Source: Anja Hauser)

Our immune system is characterized by a dynamic interplay of various cell types. Immune cells can act in relation to other immune cells, but also in relation to tissue cells. This is even necessary in order for immune reactions to set in, to continue and also to be regulated. Whenever scientists are looking to develop new therapeutic approaches, they must also understand the interplay of the various cells on a molecular basis and within a spatial context in the tissue. Traditionally, tissue samples of animals are used and examined by histological methods. Conventional histological methods, however, can only examine four parameters, or markers, per experiment. This limitation means that for further measurements further tissue sections are required. In her laboratory, Charité scientist Prof. Anja Hauser-Hankeln has established a method that facilitates the measurement of up to 50 markers – it is called multiplex microscopy (MELC). This means that fewer tissue samples are required, which has a direct effect on the total number of test animals required. In addition, the veterinarian has expanded the method to the analysis of human samples. Multiplex microscopy thus not only reduces the amount of animal testing, but also its application by replacing it. This approach also harbors a high potential for translational research, meaning the transfer of knowledge from basic research to clinical therapies.


Murine thyroid follicle organoids to assess endocrinal disruptors

The thyroid model was originally developed by scientists in Belgium and the USA and was published in “Nature” in 2012. Charité scientists have developed it further to test the influence of endocrinal disruptors on the thyroid function. A large amount of animal testing was thus avoided. (Source: Charité 3R)

The thyroid – seen as an endocrinal organ and source of the thyroid hormones – is an important regulator in the human organism, for its energy metabolism, for example. Thyroid hormones also play a decisive role in the prenatal and early infant development and maturation of the central nervous system(CNS).

Additional to a number of diseases which affect the thyroid, various industrial chemicals are suspected to influence its function as well. To examine the influence of such endocrinal disruptors, more and more organoid models are meanwhile available to complement the simple enzyme and transporter tests. These models perspectively reflect the function of the entire organ and thus possess a potentially higher physiological relevance. What’s more, they are a game-changing alternative to animal testing.

Such a system is currently adapted and further developed at the Charité Institute for Experimental Endocrinology (IEE)* under the helm of Dr. Kostja Renko. It is based on murine(originating from mice) embryonic stem cells, which the scientists then differentiate into smaller, functional sub-segments of the thyroid, so-called follicles. Only when organized in such a follicular structure, the functional cells of the thyroid, above all thyrocytes, are able to absorb iodine and produce thyroid hormones(see picture) The model is thought to be very robust and will facilitate the testing of the substances in question without additional animal testing.

*The IEE is partner in the current ATHENA project funded by the EU(Assays for the identification of Thyroid Hormone axis-disrupting chemicals)

Neuroblastoma organoids from genetically modified mice

The neuroblastoma (NB) is a dangerous cancer in infancy, with more than 60 percent of afflicted children dying from a high-risk tumor. In order to develop new therapeutic options for the little patients, various steps need to be taken before a drug can be tested in a clinical study. Many promising drugs are first tested in a 2D cell culture; later, effectiveness is tested on a living organism such as genetically modified mouse models portraying the symptoms of the cancer. Of late, there has been an increase in the application of improved cell culture models, the so-called organoid models. In these, “mini tumors” are cultivated from tumor cells in a petri dish and then undergo the same treatment as in a mouse model.

In this 3R project, Prof. Dr. Johannes Schulte and his team fashion such mini tumors from already existing neuroblastoma mouse models to test a new drug combination of Volasertib and Alisertib. The findings are then compared with those of the mouse trials that the scientists perform within the framework of the research consortium ENABLE.

It is the goal of the 3R project to demonstrate that these organoids are on par as an instrument with the mouse models that still form the standard – with equal gain in scientific insight, but a much faster experimental stage. The novel 3D neuroblastoma model has the decisive advantage of requiring much fewer test animals.

Organoids as a new test model for the neuroblastoma

New medication against the neuroblastoma may be tested on organoids in the future. This saves a large amount of animal testing.

The treatment of solid tumors in infancy is still a huge challenge, despite all improved therapies. The work group of Dr. Anton G. Henssen is trying to develop new therapies for the neuroblastoma – a tumor, which is difficult to treat in some cases and unfortunately often results in the death of the little patients. As a rule, new medication must first be tested in tumor models in the laboratory before they can be applied to patients in the course of a clinical study. Often, scientists have to rely on animal models, as no better models are available yet. Henssen and his co-scientists are looking to change just that and they are working on an alternative with the support of the 3R program: in co-operation with the clinic for infant oncology at the Charité, the scientists develop 3D models of neuroblastoma, which in time shall replace animal testing. These miniature tumors are won from the tumor tissue of the patients and grow as a gelatinous substance in a petri dish. In the models they developed, the scientists are looking to test various substances for their effectiveness against the neuroblastoma. The project’s goal is to find new therapies for children suffering from neuroblastoma and to simultaneously reduce the amount of animal testing.

RealBarrier – model for the examination of the blood-air-barrier

Animal-free alternative: the RealBarrier system accurately represents the anatomy of the blood-air-barrier. (Source: C3R)

The blood-air-barrier fulfills a vital function of the lung, as it separates the air-filled space of the lung bubbles (alveoli) from the capillary blood. The present gold standard for the examination of the barrier function of the lung is the intact organ, which usually comes from living (in vivo) or dead (ex vivo) animals. Alternative models in cell cultures do not sufficiently reflect this complex system with its manifold cell types and inter-cellular processes of communication. As of today, they are only model systems with many shortcomings. In order to successfully replace ex-vivo or in-vivo examinations of the lung barrier in the animal model, the team of Prof. Dr. Wolfgang Kübler develop an innovative RealBarrier system with the aid of Charité 3R. The promising in-vitro model combines a blood vessel system of continuous perfusion with an alveolar side exposed to air, which anatomically correctly represents the multi-cellular environment of the blood-air-barrier. RealBarrier is a versatile alternative to examine physiological and pathophysiological phenomena at the alveolar-capillary barrier. It holds the potential to replace animal testing and minimize the numbers in animal testing, it can be used in other areas of scientific interest, and may well be used by scientific institutions as well as the pharmaceutical industry.