Caloni, Francesca

Cazzaniga, Alessandra

Gutleb, Arno C

Hartung, Thomas

Kandarova, Helena

Moutaharrik, Saliha

Ranaldi, Giulia

Rashidi, Hassan

Wilflingseder, Doris

English

AIR Universita degli studi di Milano

ALTEX 41(4), 2024 666sometimes contradictory; therefore, further studies are necessary to better understand MNP effects on intestinal systems.Helena Kandarova, CEM & FChFT Bratislava, held a lecture entitled “Challenges and complexities in medical device testing in vitro: from 2D to 3D models”. The evaluation of biocompatibility is pivotal for ensuring the safety and efficacy of medical devices (Kandarova and Pôbiš, 2024). Traditional testing methods, which primarily utilize animal models and basic in vitro cytotoxicity tests as prescribed by ISO 10993, are foundational yet often inad-equate in mimicking the complex biological interactions of human tissues. This shortfall becomes increasingly significant as medical devices grow more complex, integrating various materials intend-ed for diverse application scenarios. Standard biocompatibility assessments such as cytotoxicity, irritation, and sensitization are mandated for most devices, but additional evaluations like geno-toxicity, systemic toxicity, hemocompatibility, and implantation studies may be necessary based on the device’s nature and intend-ed use. Despite these requirements, the medical device industry continues to rely heavily on animal testing, with a slower adop-tion of alternative methods compared to other sectors. The field of tissue engineering has seen significant advancements leading to the development of three-dimensional human reconstructed tis-sue models, including spheroids, organoids, and planar systems. These innovative models have been incorporated into regulatory assessments for chemicals, cosmetics, and pesticides, and are in-creasingly recognized in the medical device sector. The recent in-tegration of the 3D human epidermis model into ISO 10993-23, following successful validation trials, marks a pivotal develop-ment. The use of sophisticated in vitro models, enhanced by tech-nologies such as microfluidics and 3D printing, suggests a poten-tial shift in medical device safety assessments. These technologies enable the creation of physiologically relevant models that closely replicate human organ structures and dynamics, thus improving the accuracy of biocompatibility tests. The presentation discussed the challenges and complexities of medical device testing, but also the urgency for evolving regulatory frameworks to facilitate the adoption of these advanced methodologies, promoting more ac-curate, ethical, and scientifically rigorous biocompatibility testing. Thomas Hartung, Center for Alternatives to Animal Testing (CAAT), Baltimore, focused his talk on “Alternatives to animal testing: The now, the new, and the next” The NOW: Despite growing discomfort and doubt in its scientific value, animal test-ing remains the dominant technology in toxicology, with some replacements by rather simple cell cultures. The NEW: The ad-vent of stem cells and bioengineering has made human micro-physiological systems (MPS) available, which replicate aspects The International Fifth Virtual Summer School Lake Como School of Advanced Studies “Non-Animal Models: Complexity for Inter-actions… Connecting Science” (https://namo.lakecomoschool.org/), held on May 15-16, 2024 and chaired by Francesca Caloni, Università degli Studi di Milano, Department of Environmental Science and Policy, was focused on innovative in vitro method-ologies like 3D cultures, spheroids, organoids, and integrated mi-crofluidic perfusion systems (MPS) and their interplay with other advanced technologies, also involving ethical considerations. The Summer School was attended by young scientists from all over the world with different scientific backgrounds. Francesca Caloni gave a brief introduction. Non-animal methods have applications way beyond toxicology in multi-faceted biomedical research; however, they need to overcome multiple barriers to gain acceptance and replace animal testing (Sewell et al., 2024). Giulia Ranaldi, Food and Nutrition Research Centre, Council for Agricultural Research and Economics, CREA-AN, Rome held a presentation entitled “In vitro intestinal toxicity of micro- and nanoplastics”. Fragmentation of plastics across their life cycle produces debris and particles of mixed morphology, chemical composition, and size, including in the micron and nanometer range, defined as micro-nanoplastics (MNPs). MNPs represent an emerging class of contaminants that have been detected in almost every biosphere environment. Despite MNPs constitute a poten-tial public health concern, MNP risk assessment remains to be addressed since substantial data gaps regarding MNP classifica-tion, detection, exposure as well as toxicity have not been clearly determined. Human exposure has been demonstrated as plastic particles have been detected in human organs, blood, and in breast milk. Ingestion of MNPs from contaminated food and water rep-resents the main way of human intake, thus understanding MNP exposure and the impact of MNPs on the gastrointestinal system is fundamental to evaluate possible harmful effects. In vitro intesti-nal systems represent a valuable approach to study intestinal toxic-ity and are also used for studying MNP intestinal impact. Intestinal absorption mechanisms, toxic effects, and intestinal barrier per-turbation are under investigation using 2D intestinal differentiated in vitro cell lines, 3D intestinal organoids, as well as microfluidic systems. Although studies are still at a preliminary stage, results indicate the ability of MNPs to cross intestinal epithelia by endo- and transcytosis. Potential toxic mechanisms including oxidative stress, inflammatory challenge, and perturbation of barrier func-tion have been suggested, and the role of the different cell types residing in the intestinal mucosa has been highlighted. However, experimental protocols are relatively heterogenous, and results are Meeting ReportNon-Animal Models:  Complexity for Interactions… Connecting Sciencedoi:10.14573/altex.2407181Meeting RepoRtsALTEX 41(4), 2024 667airway epithelia from infection with different SARS-CoV-2 vari-ants of concern (Zaderer et al., 2023), influenza A and B (Zaderer et al., 2024), and (iii) corticosteroids created a suppressive micro-environment and promoted fungal invasion in epithelial/immune respiratory models (Luvanda et al., 2021). These human systems offer the opportunity to evaluate novel therapeutic intervention strategies or test drugs or vaccination efficiency in a personalized manner. Arno C. Gutleb, Environmental Sustainability Assessment and Circularity (SUSTAIN) Unit, Luxembourg Institute of Science and Technology, presented “Complex – more complex – most complex – but why?” In recent years in vitro systems have be-come increasingly complex in an attempt to better mimic potential interactions between different cell types in tissues. Such in vitro systems can consist of several cell types cultured in 3D orientation using inserts with porous membranes, membranes with a three-dimensional structure, 3D-scaffolds, and organ-on-a-chip format. Cell models can make use of cell lines, primary cells, and increas-ingly also induced pluripotent stem cells (iPSCs). Another level of complexity added to such models can come from the combination of in vitro models representing different organs or adding physical stress such as pressure, membrane tension, or change of oxygen levels. The final question, however, is what is the added value of such increasing complexity? The added value of combining dif-ferent cells into a complex 3D in vitro model has been shown for a model representing the alveolar barrier including immune cells. Gene expression data showed an increasing correlation between transcriptional profiles observed in the alveolar barrier model when adding immune cells to the simple barrier model and normal human lung tissue (Marescotti et al., 2019).Hassan Rashidi, NIHR Great Ormond Street Hospital Bio-medical Research Centre, UCL Great Ormond Street Institute of Child Health, University College London, presented a lecture en-titled “In vitro platforms to assess liver toxicity”. Drug-induced liver injury (DILI) poses a significant challenge in drug devel-opment and patient safety assessment. Non-animal models offer promising alternatives for studying DILI mechanisms and drug toxicity in a controlled and ethically acceptable manner. How-ever, the complexity of liver physiology and the intricate inter-actions between different hepatic cell types present challenges in recapitulating the dynamic microenvironment of the liver in vitro. The current landscape of non-animal models for studying DILI was discussed, including 2D and 3D cell culture systems, organ-on-a-chip platforms, and computational models. The advantages and limitations of these models in simulating hepatotoxicity, me-tabolism, and immune responses were explored, highlighting the need for multi-cellular and multi-organ interactions to accurately model DILI pathways. Additionally, recent advances in integrat-ing liver models with other organ systems, such as the gut and immune system, to better mimic systemic drug effects and inter-organ crosstalk were discussed. Finally, future directions and challenges in refining non-animal models for DILI research were addressed, including standardization of protocols, incorporation of human-relevant endpoints, and validation against clinical da-ta. Overall, non-animal models offer valuable tools for advanc-of organ architecture and function (Marx et al., 2020). They are ready for validation, but we have to adapt quality assurance and reporting as well as the validation process. The NEXT: The syn-ergy of data generation and improvement of computers and al-gorithms has increased the power of artificial intelligence (AI) more than a billion-fold since the term was coined in 1956: Data in the world double every 18 months, i.e., 90% of all data were produced in the last three years; computers double in capacity every 24 months (Moore’s law), and AI algorithms double in ca-pacity every 3 months since 2010. For most human skill tests, AI performs better than 90% of us. For toxicology and safety pharmacology, AI promises support for data retrieval, evidence integration (systematic reviews, risk assessments), predictive toxicology of untested compounds, digital pathology, and support in reporting (Hartung, 2023a). The prospects of animal replace-ment with better accuracy in (human) prediction, ethical benefits, and cost-effectiveness are enormous (Hartung, 2023b). Beyond this, accelerated assessments with automated data analyses, re-al-time monitoring, and complex analyses come into reach with user-friendly prediction tools. These changes also promise to democratize knowledge, encourage open-access databases, algo-rithms, and publications. As a copilot for toxicology, it empow-ers researchers, regulators, consumers, and industry (Kleinstreuer and Hartung, 2024). Combining brain MPS with AI, the new field of organoid intelligence (OI) (Smirnova et al., 2023) was created, which seeks to establish learning and memory-in-a-dish, with op-portunities to cover some of the most complex neurological haz-ards with functional endpoints.The lecture presented by Doris Wilflingseder, Institute of Hygiene and Medical Microbiology, Medical University of Innsbruck and University of Veterinary Medicine, Department of Pathophysiology, Vienna, was titled “Infectious disease mod-els 3.0”. The growing spread of emerging infectious diseases, such as COVID-19 or resistant pathogens, indicates the need to speed up research on repurposing already approved drugs or test-ing novel innovative compounds. Since effective drugs or vac-cines must induce both humoral and cellular responses against pathogenic challenges, novel alternative human approaches are needed including cellular and human immune components, and improved methods for delivery must be tested. Rapid develop-ments in high-content screening as well as organotypic cultures provide groundbreaking new tools to study pathogen transfer at entry sites or to test novel treatment strategies. Therefore, we de-sign optimized intelligent human barrier models combined with infection-relevant immune cells and humoral components to characterize and hinder overshooting host responses, pathogen entry, and initial transmission steps within a 3D system. These human systems offer improved power to test delivery methods, adjuvants, repurposing of drugs or novel vaccination approaches in high throughput. Using these models, we could demonstrate that: (i) C5aR inhibition of epithelial cells suppressed inflamma-tion as assessed by local complement production, anaphylatoxin, and pro-inflammatory cytokine release, and maintained epithelial integrity in SARS-CoV-2-infected primary human airway epithe-lia (Posch et al., 2021), (ii) antiviral sprays effectively shielded Meeting RepoRtsALTEX 41(4), 2024 668ing our understanding of DILI mechanisms and improving drug safety assessment, but further efforts are needed to enhance their complexity and predictive capabilities.During the Summer School, the participants were invited to ad-dress some questions related to the topics that were covered and to express their vision on adoption of NAMs in toxicology. ReferencesHartung, T. (2023a). Artificial intelligence as the new frontier in chemical risk assessment. Front Artif Intell 6, 1269932. doi: 10.3389/frai.2023.1269932 Hartung, T. (2023b). ToxAIcology – The evolving role of artificial intelligence in advancing toxicology and modernizing regula-tory science. ALTEX 40, 559-570. doi:10.14573/altex.2309191 Kandarova, H. and Pôbiš, P. (2024). The “Big Three” in biocom-patibility testing of medical devices: Implementation of alterna-tives to animal experimentation-are we there yet? Front Toxicol 5, 1337468. doi:10.3389/ftox.2023.1337468 Kleinstreuer, N. and Hartung, T. (2024). Artificial intelligence (AI) – It’s the end of the tox as we know it (and I feel fine). Arch Toxicol 98, 735-754. doi:10.1007/s00204-023-03666-2 Luvanda, M. K., Posch, W., Noureen, A. et al. (2021). Dexamethasone creates a suppressive microenvironment and promotes Aspergillus fumigatus invasion in a human 3D epi-thelial/immune respiratory model. J Fungi 7, 221. doi:10.3390/jof7030221 Marescotti, D., Serchi, T., Luettich, K. et al. (2019). Added val-ue of complexity: How complex should an in vitro model be? The experience on a 3D alveolar model. ALTEX 36, 388-402. doi:10.14573/altex.1811221 Marx, U., Akabane, T., Andersson, T. B. et al. (2020). Biolo-gy-inspired microphysiological systems to advance medicines for patient benefit and animal welfare. ALTEX 37, 364-394. doi:10.14573/altex.2001241 Posch, W., Vosper, J., Noureen, A. et al. (2021). C5aR inhibition of nonimmune cells suppresses inflammation and maintains epithelial integrity in SARS-CoV-2-infected primary human airway epithelia. J Allergy Clin Immunol 147, 2083-2097.e6. doi:10.1016/j.jaci.2021.03.038 Sewell, F., Alexander-White, C., Brescia, S. et al. (2024). New approach methodologies (NAMs): Identifying and overcom-ing hurdles to accelerated adoption. Toxicol Res 13, tfae044. doi:10.1093/toxres/tfae044 Smirnova, L., Morales Pantoja, I. E. and Hartung, T. (2023). Or-ganoid intelligence (OI) – The ultimate functionality of a brain microphysiological system. ALTEX 40, 191-203. doi:10.14573/altex.2303261 Zaderer, V., Dichtl, S., Posch, W. et al. (2023). GlyPerA™ effec-tively shields airway epithelia from SARS-CoV-2 infection and inflammatory events. Respir Res 24, 88. doi:10.1186/s12931-023-02397-3 Zaderer, V., Diem, G., Posch, W. et al. (2024). P80 natural essence spray and lozenges provide respiratory protection against Influ-enza A, B, and SARS-CoV-2. Respir Res 25, 102. doi:10.1186/s12931-024-02718-0  AcknowledgementsA special thanks to Professor Giulio Casati, Director of the Como Lake School of Advanced Study, Como, Italy and to the active participants of the Fifth Summer School.Francesca Caloni1, Alessandra Cazzaniga2,  Arno C. Gutleb3, Thomas Hartung4,5, Helena Kandarova6,7,  Giulia Ranaldi8, Hassan Rashidi9, Doris Wilflingseder10,11  and Saliha Moutaharrik121Università degli Studi di Milano, Department of Environmental Science and Policy (ESP), Milan Italy; 2Fondazione Alessandro Volta, Italy; 3Luxembourg Institute of Science and Technology, Environmental Sustainability Assessment and Circularity (SUSTAIN) Unit, Luxembourg; 4Center for Alternatives to Animal Testing, Bloomberg School of Public Health and Whiting School of Engineering, Doerenkamp-Zbinden-Chair for Evidence-based Toxicology, Johns Hopkins University, Baltimore, MD, USA; 5University of Konstanz, CAAT-Europe, Konstanz, Germany; 6Centre of Experimental Medicine (CEM), Institute of Experimental Pharmacology and Toxicology, Slovak Academy of Sciences, Bratislava, Slovakia; 7Faculty of Chemical and Food Technology (FChFT), Institute of Biochemistry and Microbiology, Slovak University of Technology (STU), Bratislava, Slovakia; 8Food and Nutrition Research Centre, Council for Agricultural Research and Economics, CREA-AN, Rome, Italy; 9NIHR Great Ormond Street Hospital Biomedical Research Centre, UCL Great Ormond Street Institute of Child Health, University College London, London, UK; 10Institute of Hygiene and Medical Microbiology, Medical University of Innsbruck, Innsbruck, Austria; 11University of Veterinary Medicine, Department of Pathophysiology, Infectiology Unit, Vienna, Austria; 12Università degli Studi di Milano, Department of Pharmaceutical Sciences (DISFARM), Section of Pharmaceutical Technology and Legislation “M.E. Sangalli”, Milan, Italy

Non-animal models: Complexity for interactions...Connecting science

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Non-animal models: Complexity for interactions...Connecting science

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