A New Way to Evaluate Chemical Safety and Assess Risk
TPI.tv: improving science through animal-free innovations and research
Introducing TPI.tv : a video platform by experts striving to improve science through animal-free innovations and research.
Monique Janssens (personal account): Why we need the Transition towards Animal-free Innovations
Why is there a Transition towards Animal-free Innovations, while we have the 3Rs, including Replacement? Well, there is a difference. Animal experiments should no longer be the golden standard of reference. We should not ask: Is this animal-free method good enough to replace animal experiments? But: What is the research question, and how do I get the best answer, preferably without animals? I know that many researchers are doing this already. But we can do even more! It’s also about involving the full chain of parties, including patients, financers, legislators and companies. That is why the transition movement works with interdisciplinary networks and Helpathons. The transition helps to innovate, to accelerate and to implement. At the same time, there is no need to throw the 3Rs overboard. Actually, we owe applying them to the lab animals of today. But by innovating we can develop even more new practices in research and education that bring about better results for science in less time and often with less costs. Without using animals.
GUTS BV - small intestine-on-a-chip and advanced computational analysis for compound and protein screening
GUTS BV is a contract research organization offering its 3-dimensional state-of-the-art small intestinal in vitro model in combination with custom computational analysis approaches. The small intestinal model was developed during Dr. Paul Jochems PhD research at Utrecht University in the group of Prof. Roos Masereeuw. In comparison to the current gold standard (Transwell model), they show improvement in cell differentiation (all major specialized cell types present), physiological structure (3D tube- and villi-like structures) and a functional epithelial barrier. After acquiring experimental data from this model computational analysis approaches are used to score and compare measured compounds for all tested biological parameters at once. The combined effort of improved in vitro modelling and data analysis is believed to result in an enhanced preclinical predictability. GUTS BV was nominated for the Venture Challenge 2021 for their development of an intestinal model combined with advanced computational analysis for protein and chemical compound screening. Research papers: https://www.sciencedirect.com/science/article/pii/S0887233318307811 https://www.mdpi.com/2072-6643/12/9/2782/htm https://www.nature.com/articles/s41538-020-00082-z LinkedIn: https://www.linkedin.com/company/71016128/
Human neuronal cell models for in vitro neurotoxicity screening and seizure liability assessment
Anke Tukker was a PhD candidate in the Neurotoxicology Research group of Dr. Remco Westerink at the Institute for Risk Assessment Sciences at Utrecht University. Dr Westerink’s research group investigates the mechanisms of action of toxic substances on a cellular and molecular level using in vitro systems. Anke's project aimed to develop a human induced pluripotent stem cell (hiPSC)-derived neuronal model for in vitro neurotoxicity screening and seizure liability assessment. Using micro-electrode arrays (MEAs), she showed that these models mimic in vivo neuronal network activity. When these hiPSC-derived neurons are mixed with hiPSC-derived astrocytes, they can be used for in vitro seizure liability assessment. Comparing these data with data obtained from the current used model of ex vivo rodent cortical cultures, she found that these human cells outperform the rodent model. Here research thus contributes towards animal-free neurotoxicity testing. Anke Tukker has won the public vote of the Hugo van Poelgeest prize 2020 for her research on human neuronal cell models for in vitro neurotoxicity screening and seizure liability assessment. Neurotoxicology Research Group, IRAS, Utrecht University: https://ntx.iras.uu.nl/NTX_at_Iras
Scientific solutions for the gap in translational medicine: skin model platform with melanoma (3D melanoma)
The developing process of a new drug, from first testing to regulatory approval and ultimately to market is a long, costly, and risky path. Noteworthy is the fact that almost 95% of the drugs that go into human trials fail. According to the National Institutes of Health (NIH), 80 to 90% of drug research projects fail before they ever get tested in humans. The value of preclinical research, mainly conducted in animal model experiments for predicting the effectiveness of therapies and treatment strategies in human trials, has remained controversial. Only 6% of the animal studies are successfully translated into the human response. Breaking down failure rates by therapeutic area, oncology disorders account for 30% of all failures. The absence of human-relevant models with receptors, proteins, and drug interactions in the in situ microenvironment leaves a gap in the scientific discovery process of new therapies. In this context, the present work presents the development of a sophisticated in vitro skin model platform focus on boosting melanoma treatment. The results showed a physiological microenvironment of human skin with epidermal differentiation and development of stratified layers (basement membrane, stratum spinosum, stratum granulosum, and stratum corneum). Furthermore, it was observed the pathophysiological microenvironment of the melanoma with invasion or migration through the basement membrane into the dermis and no epidermal differentiation. Vemurafenib treatment, the gold standard which targets BRAF mutations, showed a decrease in proliferation and invasion of melanoma tumors, with an increase in epidermis keratinization. Melanoma incidence continues to increase year-on-year and is currently responsible for >80% of skin cancer deaths. It is the most common cutaneous form and is known to have the highest mutational load of all cancers. Nowadays, patients with advanced melanoma BRAFV600E mutation can benefit from monotherapies or targeted therapies. Although the initial response rate is effective, disease progression and tumor chemoresistance rapidly occur in the majority of patients. Therefore, the treatment of melanoma remains a challenge, and despite the advances, there is still an urgent need to identify new therapeutic strategies. 3D Model Melanoma is considered one important tool for studying the evolution of the pathology, as well as evaluating the effectiveness of new therapeutic approaches.
Optimizing CAR-T-cell therapy using 3D tumor models and real-time cell imaging
Chimeric antigen receptor (CAR) T-cell therapy accounts for one of the most promising therapeutic advances in cancer immunotherapy. In this form of adoptive cell transfer, T-cells of a patient are engineered to express so-called ‘CARs’, in which the antigen-recognition capacity of antibodies is combined with T-cell activating domains. So far, CAR-T-cell therapy obtained its most impressive results in hematological malignancies resulting in the approval of five CAR-T cell products by the FDA for hematologic indications. However, CAR-T-cell therapy has not mirrored its success in solid tumors. The poor efficacy of CAR-T-cell therapy in solid tumors has, in part, been attributed to the lack of understanding in how CAR-T-cells function in a solid tumor microenvironment. Classical validation methods rely on the use of specificity and functionality assays in 2D models against adherent target cells or target cells in suspension. Yet, by using these models, observations made in vitro may differ greatly to an in vivo situation where tumors are engrafted in 3D structures. We developed a more relevant and translational 3D tumor model using eGFP+ target cells. This allows us to couple 3D tumor cell killing by CAR-T-cells to live-cell imaging, providing an efficient quantification of target cell death. As proof- of-concept, we used a 3D model of eGFP+ glioblastoma cells and CAR-T-cells targeting a pan-cancer antigen. This 3D glioblastoma model allowed us to show that classical scFv-based CAR-T-cell therapy of glioblastoma cells can be improved by nanoCAR-T-cells. Furthermore, combining nanoCAR-T-cell therapy with a genetic approach of nanobody-based anti-PD-L1 immune checkpoint blockade further increased the cytotoxicity of the nanoCAR-T-cell therapy.
Biotransformation of two proteratogenic anti-epileptics in the zebrafish (Danio rerio) embryo
The zebrafish (Danio rerio) embryo has gained interest as an alternative model for developmental toxicity testing, which still mainly relies on in vivo mammalian models (e.g., rat, rabbit). However, cytochrome P450 (CYP)-mediated drug metabolism, which is critical for the bioactivation of several proteratogens, is still under debate for this model. Therefore, we investigated the potential capacity of zebrafish embryos/larvae to bioactivate two known mammalian proteratogens, carbamazepine (CBZ) and phenytoin (PHE) into their mammalian active metabolites, carbamazepine-10,11-epoxide (E-CBZ) and 5-(4-hydroxyphenyl)-5-phenylhydantoin (HPPH), respectively. Zebrafish embryos were exposed to three concentrations (31.25, 85, and 250 μM) of CBZ and PHE from 51⁄4 to 120 hours post fertilization (hpf) at 28.5°C under a 14/10 hour light/dark cycle. For species comparison, also adult zebrafish, rat, rabbit and human liver microsomes (200 μg/ml) were exposed to 100 μM of CBZ or PHE for 240 minutes at 28.5°C. Potential formation of the mammalian metabolites was assessed in the embryo medium (48, 96, and 120 hpf); pooled (n=20) whole embryos/larvae extracts (24 and 120 hpf); and in the microsomal reaction mixtures (at 5 and 240 minutes) by targeted investigation using a UPLC–Triple Quadrupole MS system with lamotrigine (0.39 μM) as internal standard. Our study showed that zebrafish embryos metabolize CBZ to E-CBZ, but only at the end of organogenesis (from 96 hpf onwards), and no biotransformation of PHE to HPPH occurred. In contrast, our in vitro drug metabolism assay showed that adult zebrafish metabolize both compounds into their active mammalian metabolites. However, significant differences in metabolic rate were observed among the investigated species. These results highlight the importance of including the zebrafish in the in vitro drug metabolism testing battery for accurate species selection in toxicity studies.
Lung tumor spheroids for onco-immunological research
Lung cancer thrives in a complex multicellular tumor microenvironment that impacts tumor growth, metastasis, response, and resistance to therapy. While orthotopic murine lung cancer models can partly recapitulate this complexity, they do not resonate with high-throughput immunotherapeutic drug screening assays. To address the current need for relevant and easy-to-use lung tumor models, we established a protocol for fully histo-compatible murine and human lung tumor spheroids, generated by co-culturing lung fibroblasts with tumor cells in ultra-low adherence 96-well plates. Moreover, we describe their application potential to study tumor-stroma organization, T-cell motility, and infiltration as well as distinct macrophage subsets’ behavior using confocal microscopy. Finally, we report on a 3D target specific T-cell killing assay that allows spatio-temporal assessment using live cell imaging and flow cytometry. This lung tumor spheroid platform can serve as a blueprint for other solid cancer types to comply with the need for straightforward onco-immunology assays.