Workshop: Non-Animal New Approach Methodologies (NAMs) in Biomedical Research

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November 22-23, 2021, 9am-12pm EST/ 3-6pm CET

Online Workshop (via Zoom)

The Johns Hopkins University Center for Alternatives to Animal Testing (CAAT) and the Animal Protection Commissioner of Berlin are inviting early career scientists for a two-day Online Workshop focusing on animal-free New Approach Methodologies (NAMs) in biomedical research. The program will include presentations and interactive sessions. Following the call for abstracts, up to ten early career scientists will be selected to present their own research involving NAMs. The program will also include a session with established scientists from academia, industry, government, and NGOs who will answer the participants’ questions and share advice and guidance on future careers in the field of non-animal methods. In another session, we will discuss topics such as how to tackle the ‘publish or perish’ trap, and where to find funding as well as ongoing training opportunities in NAMs.
The Workshop is free of charge. Please use the form below to apply and email it to Dr. Kathrin Herrmann at . Deadline Extension until November 20, 2021 For registration a CV and a motivational letter is needed.

Agenda (all times in CET)

Monday, November 22:

15:00-15:10 Welcome and ice breaker by Kathrin Herrmann, CAAT & Animal Protection Commissioner of Berlin and Helena Hogberg, CAAT

Kathrin Herrmann

PhD, is a veterinary expert in animal welfare science, ethics and law. Since 2017, she has worked at the Center for Alternatives to Animal Testing (CAAT) at Johns Hopkins University, USA, where she directs the Beyond Classical Refinement Program. Her work addresses the reproducibility and translatability crises that science is facing. Taking into consideration insurmountable interspecies differences, solely refining animal studies will not be sufficient to advance human healthcare. Consequently, Kathrin’s Program is critically appraising current animal use practices in science. To inform the public about animal use in science and its alternatives, Kathrin initiated and co-edited the open access book Animal Experimentation: Working Towards a Paradigm Change (Brill 2019). It features 51 authors who critically review current animal use in science, present new and innovative non-animal approaches and offer a roadmap towards a human biology-based science. In November 2020, Kathrin took an additional position. She is the Animal Protection Commissioner of Berlin, Germany, a role in which she advises the government of Berlin in various animal welfare and protection issues.

Helena Hogberg

PhD, is the Deputy Director of the Center for Alternatives to Animal Testing (CAAT) at the Johns Hopkins Bloomberg School of Public Health, Baltimore, MD. She received her PhD in Toxicology from the Physiology Department at Stockholm University, Sweden in 2009. The scientific work during her Ph.D. was performed at the European Centre for the Validation of Alternative Methods (ECVAM), European Commission, Ispra, Italy and aimed to develop new in vitro approaches to detect chemicals with developmental neurotoxicity (DNT) potential with a focus on gene expression and electrical activity recordings. Together with Dr. Hartung she started the current laboratory at CAAT as a Postdoctoral Fellow in 2010. She was later promoted to faculty position and was appointed the Deputy Director of the Center in the beginning of 2016. Her current research activity is still in the field of DNT with the use of emerging tools such as 3D organotypic cell models, induced pluripotent stem cells and omics (transcriptomics and metabolomics) approaches.

15:10-16:00 Session 1: Publish or perish?

15:10-15:25 Talk by Clemens Wittwehr, European Commission’s Joint Research Centre

Clemens Wittwehr

is the competence group leader for knowledge sharing in the Chemical Safety and Alternative Methods Unit of the European Commission’s Joint Research Centre (JRC). Together with his group, Clemens explores innovative ways to increase relevant stakeholders’ awareness of the facts and information they need to increase the uptake of non-animal approaches in regulatory and biomedical domains. By combining the global application of the Adverse Outcome Pathway (AOP) Framework, the publication of standardized reporting templates at OECD level, the facilitation of international collaboration and analyzing the potential for further global harmonization of regulatory approaches, Clemens supports the creation of an environment that is open to the next paradigm shift in toxicology: the non-animal test revolution. In this context, Clemens is also co-managing the international “Science with impact in an era of information overload” initiative, which aims at addressing a series of problems associated with the traditional publishing paradigm.


There is a clear need to address a central paradox of current science systems: the increase in global scientific outputs indicated by the increased rate of papers published in scientific journals that are accessible to a broader range of scientists through world wide web infrastructures, is not matched by increased ease of uptake of science in decision-making contexts where science should have its greatest impact. There seem, instead, to be several hurdles that make it even more difficult to make meaningful connections between science and its potential impacts. Possible options for better knowledge production and dissemination were examined more closely at a June 2021 workshop titled “Science with Impact in an Era of Information Overload”, in which 40 delegates from key life sciences societies, policymakers, and publishing enterprises discussed potential ways out of the publication tsunami dilemma. This talk will give an overview of the problems and solutions identified, as well as the major follow-up actions that were recommended. The observations will focus on the domains of publication, interdisciplinary collaboration and science impact. Any results of the discussion following the talk will be fed back into the planning process of the initiative, which will make sure that the thoughts of early career scientists will have an impact on further steps.

15:25-15:40 Talk by Catharine E. Krebs, PhD, Physicians Committee for Responsible (PCRM), Confronting animal reliance bias in publishing

Catharine E. Krebs

PhD, is a medical research specialist with the Physicians Committee for Responsible Medicine, a nationwide organization of physicians and laypersons that promotes preventive medicine, conducts clinical research, and encourages higher standards for ethics and effectiveness in research and medical training. At the Physicians Committee, one of Dr. Krebs’ main goals is to push the National Institutes of Health (NIH), the world’s largest funder of biomedical research, toward the most responsible and effective biomedical practices in numerous areas including Alzheimer’s disease, mental health, and translational sciences. She also leads a project to improve biomedical research publishing standards, which aims to encourage more rigorous editorial oversight and peer review to prevent the publication of work that fails to meet basic ethical principles and to combat bias against innovative non-animal methods.

Abstract: Confronting animal reliance bias in publishing

Publication of research is a necessary step in the dissemination and implementation of biomedical advances and plays a central role in the advancement of researchers’ careers. Through the manuscript review process, editors and peer reviewers act as gatekeepers of biomedical research. This process is not unbiased, though. During the review of biomedical manuscripts, animal-based validation of findings, although not always justified, is often requested or required. To assess this phenomenon, which we call “animal reliance bias in publishing,” we conducted a qualitative survey investigating the experiences and perceptions of researchers and reviewers. We describe evidence that animal reliance bias in publishing exists and can be based on editors’ and reviewers’ preference, familiarity, or narrow expertise. This leads to the evaluation of manuscripts not on merit (e.g., physiological relevance of the experimental model) but on irrelevant characteristics (e.g., a reviewer’s preference for animal experiments), and contributes to the performance animal experiments for the sole purpose of appeasing biased editors and reviewers. Furthermore, this bias stands in the way of innovation and uptake of more physiologically relevant approaches such as human cell-based microphysiological systems and organoids, which have many advantages over the use of animals. Concrete steps to combat animal reliance bias in publishing may include broadening non-animal experimental model expertise on editorial boards and in reviewer pools, conducting anti-bias training, and establishing open peer review. Ultimately, a major shift in the perception of non-animal methods will be required for the advancement of ethical, effective biomedical research.

15:40-16:00 Panel Discussion with Clemens Wittwehr and Catharine Krebs

16:00-17:40 Session 2: Early Career Scientist share their research using NAMs

16:00-16:10 Julia Kühnlenz (Bayer CropScience Sophia Antipolis)

Abstract: A microfluidic thyroid-liver platform to evaluate chemical-mediated changes in the thyroid hormone system for humans

Thyroid hormones are central drivers of early development and metabolism. Recent regulatory developments demand a stricter investigation of putative chemical-mediated effects on the thyroid hormone system. In rodent studies one readout parameter are changes in the thyroid hormone levels, which can hardly be tackled by current in vitro assays. The human relevance of rodent-derived findings remains questionable. For this reason, we present a first proof-of-principle of a liver-thyroid coculture of the rat and human (1) to investigate species-differences, (2) to evaluate liver-mediated and thyroid-directed thyroid toxicities and (3) to implement physiological readouts in vitro. Our established thyroid models, thyroid follicles isolated from primary tissue, produced thyroid hormones over 21 days and presented the characteristic in vivo-like follicular architecture. Applying the thyroid hormone secretion as a readout, the TPO-inhibitory effect of the reference compound methimazole could be simulated by our models. The liver models, 3D spheroids derived from the human HepaRG cell line or primary rat hepatocytes, demonstrated functional hepatic features e.g., albumin production and responded to phase I enzyme inducers in a species-specific manner. The rat model reproduced in vivo known increases of the hepatic thyroid hormone catabolism after the treatment with e.g., beta-naphthoflavone. These inductive effects were absent in the human model. To develop a system for detecting thyroid- and liver-mediated aberrations of the thyroid hormone system, we established a co-culture of the organ models in a microfluidic 2-organ-chip device. Similar functionalities as in single cultures were demonstrated. Most notably, we reproduced in a first feasibility study the detection of thyroid- and liver-directed thyroid hormone disruptions within the rat co-culture. This work demonstrates a first step towards a more physiologically relevant system to increase the precision of extrapolating rat-derived in vivo data to the human outcome and provide an animal-free test platform for the human risk assessment.

16:10-16:20 Sabrina Madiedo-Podvrsan (Université de Technologie de Compiègne)

Abstract: Developing and investigating a new in vitro hepato-pulmonary coculture model for the toxicological study of inhaled xenobiotic

Urbanization and globalization are multiplying and complexifying pollution sources. Human exposure is acute and chronic, especially regarding widespread airborne particles. The socio-sanitary need to investigate associated risks to human health has propelled respiratory toxicology to the forefront. Meanwhile, current European research policies require to place animal welfare and predictive relevancy at the heart of scientific investigations. In this context, we describe a newly developed Lung/Liver (LuLi) in vitro coculture model which aims to recreate inter-organ interactions between a pulmonary barrier (route of entry of inhaled xenobiotics) and a detoxification organ such as the liver. The LuLi platform was characterized using acetaminophen (APAP) xenobiotic exposures to emphasize the passage and circulation of a model substance throughout the device. Two kinds of LuLi models are developed: a developmental model which allows the technical setup of the coculture platform, and a physiological model which better approximates a human vivo situation. Following 72-hour exposures, both compartments metabolically respond to the imposed stress while remaining viable and functionalized. The developmental model highlights the presence of a relevantly active and functional lung and liver crosstalk: APAP has notable adverse effects on both compartments, which tend to be reduced under coculture conditions. This phenomenon could be linked to a detoxifying activity as the xenobiotic hepatic metabolism increases. The lung/liver crosstalk’s sensibility is being investigated thanks to the physiological model: despite low exposure concentrations, APAP metabolites continue to be detected by mass spectrometry within the circuit. This approach could be an alternative in vitro method to consider possible modulation mechanisms associated with systemic toxicity and could empower predictability accuracy within the framework of the toxicokinetic evaluation of inhaled substances. Implementing such a systems toxicology approach would be a valuable tool to build more knowledge towards better hazard and risk management with regards to increasing global exposure to atmospheric pollution

16:20-16:30 Melanie Ort (Charite Universitätsmedizin und FU Berlin)

Abstract: A bone and bone marrow chip? The struggle and success of in vitro models

In vitro models and 3D systems are a great success, if used correctly and performed well, it can depict human biology more precisely than any non-human animals’ biology. Here I present a bone and bone marrow like organ-on-a-chip system, which is xeno-free and exclusively made up from isolated primary patient material. Bone cells such as osteoblasts and mesenchymal stromal cells are seeded onto a human bone scaffold and perfused in a 3D microfluidic chip. During culture cells are differentiated to osteoclasts and bone marrow immune cells and stem cells are added to the system. This unique set-up can run for weeks, enabling the study of long-term effects on bone and bone marrow (e.g. the integration of metals particles and ions into bone and tissue). The system lets us study bone turnover and remodelling and helps us identify soluble and cellular markers important in bone and bone marrow homeostasis. Despite all the advantages of the system some limitations remain. Stem cells are not properly maintained, some cell types outcompete others over time for nutrients and growth factors. The fine-tuned balance of each cell and each molecule is still a puzzling event, so more set-ups must be tested, and the system stress tested for robustness. It is necessary to adjust the culture to the research question but always benchmarking it to the patients’ reality and checking with clinicians if an outcome was correctly predicted or matches a clinical problem. With patients centred research and in vitro models we can develop a powerful tool for translational research.

16:30-16:40 Q/A
16:40-16:50 Coffee Break
16:50-17:00 Johanna Berg (TU Berlin)

Abstract: Bioprinting of tissue models for biomedical research

Bioprinting is a rapidly developing technology that enables the exact positioning of living cells embedded in biomaterials in precise spatial arrangements to fabricate engineered tissues and organs. While the ultimate goal of bio¬printing approaches is to produce organs for transplantation purposes, bioprinted tissue or organ models also hold great potential for research purposes to serve as alternatives to animal experiments. By using human cells, humanized tissue models can be generated that may produce more relevant results for human (patho-)physiology than animal models. Depending on factors such as cell type, complexity, resolution or application, there are different technologies and polymers available to generate bioprinted tissue models. Here we will focus on bioprinted lung and liver tissue models produced by pneumatic microextrusion. Following initial characterization of the models, they were used for infection studies with the influenza A virus (lung model) or human adenovirus type 5 (AdV5). Moreover, the talk will give brief insight into further challenges of the field, namely the implementation of vascularization as well as the use of numerous hidden animal components in tissue engineering approaches.

17:00-17:10 Donjetë Simnica (UK Halle)

Abstract: Responsiveness to Immune Checkpoint Inhibitors Is Associated With a Peripheral Blood T-Cell Signature in Metastatic Castration-Resistant Prostate Cancer

Purpose: Although most patients with microsatellite instable (MSI) metastatic castration-resistant prostate cancer (mCRPC) respond to immune checkpoint blockade (ICB), only a small subset of patients with microsatellite stable (MSS) tumors have similar benefit. Biomarkers defining ICB-susceptible subsets of patients with MSS mCRPC are urgently needed. Methods: Using next-generation T-cell repertoire sequencing, we explored immune signatures in 54 patients with MSS and MSI mCRPC who were treated with or without ICB. We defined subset-specific immune metrics as well as T-cell clusters and correlated the signatures with treatment benefit. Results: Consistent overlaps between tumor and peripheral T-cell repertoires suggested that blood was an informative material to identify relevant T-cell signatures. We found considerably higher blood T-cell richness and diversity and more shared T-cell clusters with low generation probability (pGen) in MSI versus MSS mCRPC, potentially reflecting more complex T-cell responses because of a greater neoepitope load in the MSI subset. Interestingly, patients with MSS mCRPC with shared low pGen T-cell clusters showed significantly better outcomes with ICB, but not with other treatments, compared with patients without such clusters. Blood clearance of T-cell clusters on ICB treatment initiation seemed to be compatible with T-cell migration to the primary tumor or metastatic sites during the process of clonal replacement as described for other tumors receiving ICB. Conclusion: The MSI mCRPC subset shows a distinct T-cell signature that can be detected in blood. This signature points to immune parameters that could help identify a subset of patients with MSS mCRPC who may have an increased likelihood of responding to ICB or to combination approaches including ICB.

17:10-17:20 Iqra Pervaiz (Texas Tech University of Health Sciences)

Abstract: Modeling Glut1 Deficiency Syndrome at the Human Blood-Brain Barrier In Vitro Using CRISPR-Cas9 Edited Induced Pluripotent Stem Cells

Background: Glucose represents one of the major energy sources for the central nervous system (CNS). Glucose uptake is mostly occurring across the blood-brain barrier (BBB), via the involvement of glucose transporters (GLUTs). At the BBB, such transport is mostly mediated via GLUT1 also known as solute carrier family 2, facilitated glucose transporter member 1 (SLC2A1). In addition to the importance of glucose for neuron metabolism, there is growing evidence that brain microvascular endothelial cells (BMECs) lining the BBB are highly glycolytic. Yet our current understanding of glucose metabolism at the BBB remains limited. GLUT1 deficiency syndrome (G1D) is an autosomal dominant childhood form of epilepsy affecting GLUT1 activity. It is often characterized by early onset of seizures, gait disorders, and intellectual disability. A major issue is the lack of a robust in vitro model of G1D to understand the pathophysiology of the BBB. The aim of this study is to develop and characterize G1D-BMECs by gene editing using induced pluripotent stem cells (iPSCs). Methods: Heterozygous SLC2A1+/- iPSC (IMR90)-c4 were produced by CRISPR-Cas9 method. Such iPSCs were differentiated into BMECs using established protocols. Cell phenotyping, barrier function, glucose uptake, and metabolism were assessed in the system. Results: SLC2A1+/- iPSCs were viable and showed no significant differences in terms of phenotype or growth, compared to unedited iPSCs. Differentiated SLC2A1+/- BMECs showed similar SLC2A1 expression, at mRNA level to the control, but showed reduced GLUT1 expression at the protein level, as well as reduced glucose uptake. A decreased barrier function was observed in these cells, without any major changes in tight junction complex and glucose metabolism, as well as impaired angiogenesis. Discussion: Our data suggest that G1D-BMECs display reduced GLUT1 expression and glucose uptake, reduced barrier tightness, and angiogenesis thus, recapitulating findings documented in vivo. Taken together, our data suggest that G1D-BMECs can be a potential tool to model G1D at the BBB in vitro.

17:20-17:30 Maren Schenke (University of Veterinary Medicine Hannover)

Abstract: Development of a universally applicable alternative method for the potency determination of Botulinum neurotoxins

Botulinum neurotoxins (BoNTs) are bacterial toxins that inhibit neurotransmission especially in motor neurons (MNs), resulting in a potentially lethal flaccid paralysis. The toxin serotypes BoNT/A1 and B1 are used for medical and aesthetic applications, for which the potency estimation of every produced batch is a prerequisite. Even though several alternative methods have been developed, the number of mice used annually is still as high as 600,000. This is due to the limitations of these alternative methods: they are often proprietary and only applicable to single products. Therefore, human MNs, the most physiologically relevant target cell type, were generated from induced pluripotent stem cells to form the basis of a universally applicable in vitro assay. The differentiated neurons were then analyzed for their suitability to be used in the potency estimation of BoNTs. For this, the cleavage of the proteins targeted by the different BoNT serotypes was quantified via Western Blot. Human MNs showed high sensitivity to BoNT/A1 and high accordance to data from mouse lethality assays. MNs were less sensitive to BoNT/B1, which is in line with species differences between humans and rodents in the sensitivity for this serotype. To conclude, MNs can be used for potency estimation of BoNT/A1 and B1 and are a physiologically relevant and sensitive model, which could vastly improve current methods for determination of BoNT activity.

17:30-17:40 Q/A
17:40-18:00 Breakout groups to prepare questions for Session 5 in day 2

Tuesday, November 23

15:00-15:10 Welcome and ice breaker by Kathrin Herrmann, CAAT & Animal Protection Commissioner of Berlin and Helena Hogberg, CAAT

15:10-16:00 Session 3: Funding and training in the field of alternatives

15:10-15:25 Talk by Adelaide Dura (JRC), European Commission’s Joint Research Centre

Adelaide Dura

is a science communication specialist at the European Commission’s Joint Research Centre that I joined in 2016 support EURL ECVAM in promoting alternatives to animal testing. I am involved in communication, dissemination and education and training projects. In addition I am currently coordinating the ECVAM Stakeholder Forum (ESTAF) and a co-editor of the annual EURL ECVAM Status Report.


Adelaide will present some training and career opportunities for early career scientists offered by the European Commission. Among them is the JRC Summer School on non-animal approaches in science that is specifically tailored for scientists with an interest in New Approach Methodologies (NAMs), organized by the Joint Research Centre every two years. In addition, there are traineeship opportunities that are available at the European Commission throughout the year. They are great for gaining experience both in the policy and research fields.

15:25-15:40 Talk by Janine McCarthy, Physicians Committee for Responsible Medicine

Janine McCarthy

is a research policy specialist with the Physicians Committee for Responsible Medicine, a nonprofit organization in Washington DC, where she works to promote the development and implementation of non-animal research methods for biomedical science and regulatory testing. Ms. McCarthy launched the Early-Career Researchers Advancing 21st Century Science, or ERA21 program, which engages the next generation of scientists and connects them to the benefits of human-relevant research. In this role, develops specialized resources for researchers, organizes workshops, educational symposia, and trainings for scientists, and engages government agencies to increase the funding of non-animal research methods to support early-career researchers. Ms. McCarthy has a Master of Public Health from the University of Albany, SUNY, and a bachelor’s degree in human development.


The advancement of new approach methodologies (NAMs) has led to fundamental improvements in human disease modeling and drug development. However, minimal funding and opportunities exist for using human biology-based models for biomedical research. Understanding the importance of early-stage scientific mentorship, the Physicians Committee designed the Early-Career Researchers Advancing 21st Century Science (ERA21) program, to speed up progress in ethical and effective scientific research by creating a new generation of scientists who utilize and champion non-animal methods of research. ERA21 aims to increase the understanding of the scientific benefits of using NAMs, educate emerging scientists about the wide range of modern, human-relevant methods available, and connect them with opportunities in this field. ERA21 offers programs to reach early-career scientists and launch promising professions, activities include seminar series, hands-on training and interactive learning, travel and presentation awards, a monthly newsletter as well as social media groups for networking and information sharing, and more.

15:40-16:00 Panel Discussion with Adelaide Dura, Janine McCarthy, Helena Hogberg & Kathrin Herrmann

Session 4: Early Career Scientist share their research using NAMs

16:00-16:10 Caroline Frädrich (Charité-Universitätsmedizin Berlin)


Background: Development and application of novel drugs frequently fail due to adverse changes of TH-concentration or thyroid histology in animal experiments. Such effects potentially arise from interference with thyroid gland function or key regulators of local thyroid hormone (TH) transport, metabolism, and action. Deiodinases are key enzymes in TH in-/activation, with impact on development, cell differentiation and energy metabolism among other physiological processes, and have therefore been identified as molecular initiating events (MIE) in the assessment of chemicals for endocrine disruption. Method: A robust semi-automatic 384-well High Throughput Screening (HTS) platform was developed and now employed for screening substances interfering with DIO2 activity, utilizing the non-radioactive Sandell-Kolthoff (SK) reaction to determine DIO-depending iodide release. In an HTS using recombinantly expressed human DIO2, 22880 compounds including 1759 FDA-approved drugs were tested at a single concentration of 20 µM. Results: At the given concentration, 4.32% of the tested drugs showed DIO2 inhibition by >25% and 0.13 % an inhibition by >50%. 19.05% of the screened compounds had characteristics of either potential DIO2 activators or SK interference. Among the inactive compounds were some well-known DIO1-selective inhibitors such as PTU or Genistein, a finding which supports the specific and predictive quality of this HTS approach. Within the screened FDA-approved drugs, 1.1% of the tested drugs showed DIO2 inhibition by >25% and 0.2 % an inhibition by >50%. Conclusion & Outlook: In this study, we were able to demonstrate that even a library of FDA approved drugs does contain compounds that may exert adverse effects on TH metabolism by DIO2 inhibition. In perspective, such compounds should be revised regarding potential side effects that might appear from such interference, e.g., abnormal patterning of TH metabolites in serum or defects that might appear from local suppression of DIO2 activity. While this screening setup is appropriate to detect endocrine disruptors (ED) affecting DIO2 activity, it does, on the other hand, also open the perspective to identify highly potent drug candidates and reference compounds for ED research. In summary, this study represents a major success in the development of an in vitro strategy to identify endocrine disruptors, characterized by inhibition of deiodinases. The established 384-well assay protocol also provides the basis for further testing of large chemical libraries against DIO2, and can be adapted to other deiodinating enzymes modulating TH availability, e.g., DIO1 or dehalogenase (IYD). Furthermore, generated data pools provide the basis to select Quantitative Structure Activity Relationship training sets to develop predictive in silico tools for preselection and toxicological assessment. Supported by ATHENA EU-Grant nº825161.

16:10-16:20 Rajas Sane (Charité-Universitätsmedizin Berlin)

Abstract: High Throughput Screening assay for large compound libraries altering DIO1 and DIO2 activity*

The importance of new functional and local endpoints is highly appreciated in the assessment of various metabolic diseases, toxicity studies and attempts to reduce the burden on animal testing. Validated in-vitro methods to assess novel endocrine targets are scarce and demands our attention. Deiodinases (DIOs) are involved in the maintenance of the local thyroid concentrations by catalyzing the activation and inactivation of thyroid hormones and have a significant impact of cell growth, development, differentiation and energy metabolism. Therefore, DIO isoenzymes have been identified as attractive endpoints in disease modelling and toxicity studies. A robust, non-radioactive High-Throughput Screening (HTS) platform was applied for identifying compounds affecting the DIO activity by determining the DIO-catalyzed iodide release detected by the Sandell-Kolthoff reaction. The human recombinant (hr) DIO1 and DIO2 were tested in absence or presence of small molecule compounds or FDA-approved drugs. In a previous study, 1953 compounds at high concentrations were screened using a hr-HEK293-DIO1 preparation in a 96 well-plate format. 22,880 compounds were screened using a hr-HEK293-DIO2 preparation in a 384well-plate format. Of the 1953 compounds tested, 38 compounds (~2%) inhibited DIO1 activity by >50%. Of the 22,880 compounds tested, 30 compounds (~0.13%) inhibited DIO2 activity by >50%. We established a robust in-vitro HTS assay for screening of compound libraries that may exert potent effects on the endocrine thyroid hormone axis. The HTS platform will be further optimized for DIO1 in a 384 well-plate format. The most potent compounds identified from the respective DIO1 and DIO2 HTS will be further validated to determine the isoenzyme specificity, IC50 values and mechanism of action. This work shows an effort towards developing in-vitro screens thereby reducing the burden on animal testing. QSAR (Qualitative Structural Analysis Relationships) training sets for in-silico toxicity predictions can also be developed from the screening results simplifying the drug discovery process.

16:20-16:30 Julia Moradi (TU-Berlin)

Abstract: Towards cruelty-free cell culture – Replacing fetal bovine serum in cell culture media for multicellular organ models

While the development of 3D printed humanized organ models is holding the prospect to produce more applicable results in biomedical studies than animal models, thus promising to make animal testing unnecessary, it often contains hidden animal suffering in the form of animal derived materials like Matrigel and the habitual addition of 10 % fetal bovine serum (FBS). Culturing human or other non-bovine mammalian cells with FBS is a frequent source of unwanted variations in experimental results as well as ethical and increasing financial concerns. Although by now this problem is met with a growing number of cell culture products ranging from humanized media additives to completely serum free media (SFM) specific for certain cell lines, the composition of these SFM is often kept secret and may be subject to change. Additionally, SFM are often not able to support the growth of more than one cell type at the same time. With my bachelor thesis I was able to show that, at a concentration of only 2.5 % the media additives manufactured from expired human blood donations, human serum (HS) and human platelet lysate (hPL), could be used instead of FBS as complex media additives to support stable growth of fibroblast/epithelial cell coculture within a 3D printed humanized lung model. However, since HS and hPL share the complex and ill-defined composition of FBS, this adaptation marks only an intermediate step towards finding a suitable defined SFM. Our research groups’ most recent project aims to adapt several often-used mammalian cell lines to FBS free conditions. The benefits of abolishing FBS go beyond the possible reduction of animal suffering: Increased control over culture conditions, better reproducibility of experimental results and, with a projected annual growth in price per liter of FBS of more than 5 %, a significant reduction in costs.

16:30-16:40 Q/A

16:40-16:50 Coffee Break

16:50-17:50 Session 5: Advice and guidance on future careers in the field of non-animal methods

Breakout groups with established scientists holding positions in academia, industry, NGOs and government

Wesley Anderson

is a research scientist at AxoSim a CRO located in New Orleans, LA, USA. He is working on the development of 3D neural tissue models and analysis techniques, including AxoSim’s proprietary BrainSim® and NerveSim® platforms. He graduated from the University of Central Florida with a PhD in Biomedical Sciences, focusing on the development of an in vitro functional 3D model of the peripheral nerve. He specializes in tissue engineering and regenerative medicine as well as neuroscience.

Thomas Hartung

MD, PhD, is the Doerenkamp-Zbinden-Chair for Evidence-based Toxicology in the Department of Environmental Health and Engineering at Johns Hopkins Bloomberg School of Public Health, Baltimore, with a joint appointment at the Whiting School of Engineering. He also holds a joint appointment for Molecular Microbiology and Immunology at the Bloomberg School. He is adjunct affiliate professor at Georgetown University, Washington D.C.. In addition, he holds a joint appointment as Professor for Pharmacology and Toxicology at University of Konstanz, Germany; he also is Director of Centers for Alternatives to Animal Testing (CAAT, of both universities. He is Chief Editor of Frontiers in Artificial Intelligence. He is the former Head of the European Commission’s Center for the Validation of Alternative Methods (ECVAM), Ispra, Italy, and has authored more than 600 scientific publications (h-index 99).

Sarah Hedtrich

obtained her PhD in Pharmacology & Toxicology from the Freie Universität Berlin in Germany in 2009. During her postdoc, she moved to the Ludwig-Maximilians-University in Munich and Tufts University in Boston, USA. After returning to Berlin 2013, she headed a junior research group and was appointed an assistant professor in 2015. She relocated her lab to the U of British Columbia in 2019 and was recently appointed as a Johanna-Quandt Professor for Translational Organ Models at the Berlin Institute of Health at Charite Berlin. She co-/authored over 70 peer-reviewed journal articles in high impact journals including the Journal of Controlled Release, Small, Nature Reviews Materials, and Theranostics. Her research focuses on inflammatory and genetic diseases of human epithelia, the development of novel therapeutic approaches and tissue engineering of complex, human-based organ models aiming for valid and predictive test systems for preclinical and fundamental research.

Lindsay Marshall

PhD, joined the Humane Society International (HSI) and the Humane Society of the United States (HSUS) in 2016 and is a Biomedical Science Advisor. Her overarching goal in this post is the encouragement of animal replacement in biomedical research and she is specifically involved in identifying alternatives to animal models for investigating human disease mechanisms and treatments. Prior to this, she was a Senior Lecturer in Immunology at Aston University in the UK, where her research was focused on the theme of human respiratory defences, and she developed multi-cellular, in vitro models of human airways. She has a PhD in Biochemistry and Cell Physiology, a Masters in Experimental and Molecular Pathology, she is a Fellow of the Royal Society of Biology and a Senior Fellow of the Higher Education Academy.

Francesca Pistollato

PhD, currently works at the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) Joint Research Centre in Ispra (Italy), where she develops and supports the use of alternatives to animal testing in toxicology and regulatory testing and in biomedical research. She previously worked at the Physicians Committee for Responsible Medicine (PCRM), Washington DC, advocating for the use of a holistic, human-based approach for Alzheimer’s disease research. Dr. Pistollato holds a master degree in nutrition and dietetics and published several works on the role of nutrition and lifestyle-related factors in the prevention of chronic degenerative diseases, such as Alzheimer’s and dementia.

Rebecca Ram

is an independent scientific research consultant with a MSc in Toxicology and BSc in Applied Biology. After a decade working in clinical trials in the pharmaceutical industry, she changed paths to focus on alternatives to animal testing and the promotion of human-relevant research, as well as some continued clinical projects, for example the 100,000 Genomes Project. Rebecca has worked for a number of scientific and animal protection organisations, most recently, the Lush Prize and Safer Medicines Trust.

Stefan Schildknecht

PhD, is Professor at the Sigmaringen University of Applied Sciences, Germany. He was previous research group leader at the Chair of In vitro Toxicology and Biomedicine at University of Konstanz. His research uses cell based assays as alternatives for animal experiments for testing of pharmacological and toxicological compounds. His research is evaluating the posttranslational oxidative modifications in proteins as readouts for disease progression predictions with a focus on Parkinson’s disease. He is also developing Adverse Outcome Pathways (AOPs) and is co-author of one OECD approved AOP describing mitochondrial dysfunction and neurotoxicity that leads to parkinsonian motor deficits.

17:50-18:00 Final remarks

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