Respiratory toxicity using in vitro methods
The airways form a barrier for inhaled compounds, however, such compounds may cause local effects in the airways or may lead to lung diseases, such as fibrosis or COPD. Cell models of the respiratory tract, cultured at the air-liquid-interface (ALI) are a relevant model to assess the effects of inhaled compounds on the airways. Such models allow human relevant exposure, which is via the air, and assessment of effects on the epithelial cell layer. At RIVM we use air-liquid-interface cultured cell models and expose these to airborne compounds to assess the effects of agents such as nanomaterials, air pollutants or compounds from cigarette smoke. By using a mechanism-based approach to assess the effects of these compounds we invest in animal-free alternatives that better predict adverse effects in humans.
Setting up a PDXO platform of pancreatic cancer with spatial -omics characterization
Pancreatic ductal adenocarcinoma (PDAC) is known for its aggressive biology and lethality. Due to a low success rate of current diagnostic and therapeutic approaches in clinic, there is an urgent need for preclinical research studies to investigate the underlying biology of this malignancy. This knowledge is indispensable to facilitate the development and validation of potential new therapeutic compounds. Superior to conventional biomedical research models, the focus of this study is on the development and use of a well-established patient-derived 3D in vivo model, mimicking the tumor as it is present in a human body. The development and characterization of pancreatic cancer derived organoids. This model is extensively analysed using advanced histological methods omics technology to perform tumor subtyping. 15 established PDAC organoid lines and their corresponding parental tumors are validated using immunostainings and DNA hotspot sequencing. This study is the first to show in situ detection of important driver mutations of pancreatic cancer, like KrasG12D, both in parental tumor and organoids. Additionally, specific culture conditions are defined to develop subtype-specific organoids which are validated using multiplex RNA in situ hybridization and transcriptomics. We are proud to collaborate in a fruitful international project, aiming to set-up a pre-clinical screening platform for pancreatic cancer based on patient-derived organoids -and xenografts. Altogether, spatial-omics in depth analysis of both models will demonstrate (1) high resemblance to parental tissue and (2) subtype-specific signatures associated with type of model. Ultimately, the screening platform can be used by pharmaceutical companies to facilitate oncological drug testing in a subtype specific way. Publications Ilse Rooman's lab: https://pubmed.ncbi.nlm.nih.gov/34330784/ https://pubmed.ncbi.nlm.nih.gov/31161208/
A Decision Support System to Investigate the Medical Management of Ventricular Assist Device Patients
Despite the positive outcomes of ventricular assist device (VAD) therapy, there are still some adverse events, such as suction, caused by the mismatch of the preload of the left ventricle and the flow of the pump. Ventricular suction can be due to multiple underlying pathophysiological conditions. However, the medical management of such patients is still not well defined. In order to analyse the potential of different therapeutic interventions to mitigate suction in different pathophysiological conditions, a suction module is embedded in a cardiovascular hybrid (hydraulic-computational) simulator. The suction module mimics the ventricular apex and its connection with a HeartWare HVAD system (Medtronic). With such a set-up, the medical management of different pathophysiological conditions can be evaluated, without the use of animals. Also watch the video on the cardiovascular simulator used for this research: https://tpi.tv/watch/76 Contact: https://www.linkedin.com/in/maria-rocchi-9aa7b4162/
In vitro predictive models of particle-induced granulomas
Léa Hiéronimus is a PhD student at the Louvain centre for Toxicology and Applied Pharmacology (LTAP, UCLouvain, Belgium). Léa is working in François Huaux's team, where we are trying to better understand how certain inhaled particles exert their toxicity. The goal is to better diagnose and treat individuals exposed to particles, but also to identify the particle characteristics which induce, or do not induce, toxic effects. For this, Léa studies a very particular cell type which seems to be involved in particle responses. Indeed, we have found the specific accumulation of the innate subset of B-lymphocytes called “B-1 lymphocytes”, which occurred during granuloma formation/maturation induced by inhaled particles in mice. According to the literature, this accumulation can be attributed to their migration from mesothelial cavities such as the peritoneum, acting as a reservoir. In addition to conventional particles-induced granulomas, which formation rely on macrophages responses, we developed new models relying on B-1 lymphocytes. Indeed, B-1 lymphocytes show a unique clustering property, that is not observed using macrophages or other subsets of B-lymphocytes (conventional B-2 lymphocytes) as purified B-1 lymphocytes regroup granuloma-inducing particles (carbon nanotubes CNT7, crocidolite asbestos, micrometric silica MinUSil and MSS, cobalt oxide,…) but not carbon black, a particle not-inducing granuloma in vivo. Additionally, we developed a model aiming to recapitulate the lung after B-1 lymphocytes migration and found that macrophages and epithelial cells (MHS and LA4 cell lines) where grouped to form spheroids when in coculture with B-1 and not B-2 lymphocytes. These models will serve as tools to identify new mediators of granuloma formation, which could serve as biomarkers and/or therapeutic targets for exposed individuals. On the other hand, we aim to propose new bioassays for the prediction of granuloma-inducing materials using alternative models. Lab website: https://uclouvain.be/en/research-institutes/irec/ltap Contact: firstname.lastname@example.org
A phytochemical assessment of the antioxidant and cytotoxic properties of Helleborus odorus subsp. cyclophyllus extract
Helleborus sp. is a member of the Ranunculaceae family, and are small, perennial herbs common in Central and Southern Europe and Asia. Their distribution in Europe elevated their position in therapeutic remedies since the ancient time and mythology. Due to their potent and rich extracts from their roots, hellebores have been used in traditional and folklore remedies as they present rich sources in glycosides. Mainly, these plants have exhibited cathartic, anthelmintic and other beneficial aspects to treat diseases, however, hellebores have also been known for their adverse and poisonous aspects. It is also because of their cytotoxic aspect that these species have also been explored as alternative approaches to cancer treatment and are mainly reported as sporadic patient cases in literature. In this study, we first focused on the phytochemical characterisation of Helleborus odorus subsp. cyclophyllus combining biochemical assays and a detailed characterisation of its antioxidant and antibacterial properties. Furthermore, regarding its toxic potential, we explored the cytotoxic toxic properties and the mechanisms of toxicity mediated effects using in vitro cell systems primary human aortic endothelial cells (HAECs). HAECs are useful for studying vascular diseases such as thrombosis, atherosclerosis, and hypertension as well as for stent-graft compatibility testing and within the 3Rs principles, avoiding animals in these studies. Results showed the cytotoxic and reactive oxygen species potential of Helleborus extract in dose and time dependent manner. Further investigation (not shown here) revealed more mechanistic effects relevant to inhibition of proliferation. Contact: https://www.researchgate.net/profile/Anna-Michalaki
Liquid marbles: a cost-effective platform to generate cardiospheres from co-cultured cardiomyocytes and cardiac fibroblast for disease modelling
Advances in three-dimensional (3D) culture techniques have shown several advantages over 2D cultures, especially by more accurately mimicking the in vivo environment. This has led to improved reproducibility and reliability of experimental results, which are important criteria in disease modelling and toxicity testing. Induced pluripotent stem cells (iPSC) provide an unlimited source for the derivation of all cell types of the adult body, including cardiomyocytes. To improve the current culture methods for multicellular cardiac spheroids, such as the hanging drop method, we explored the use of hydrophobic powders. Fumed silica nanoparticles can be used to encapsulate liquid drops, which could serve as a microenvironment for cell cultures. This microbioreactor stimulates cell coalescence and 3D aggregation while providing optimal gas exchange between the interior and the surrounding environment. Moreover, the properties of liquid marble microbioreactors render them ideal for co-culture experiments. This liquid marble technique has been previously explored and optimized for other cell types. Here we describe a protocol that allows for the derivation of functional cardiac mini organoids, consisting of co-cultured cardiomyocytes and cardiac fibroblasts. These cardiospheres can be valuable for modelling cardiac diseases in vitro and assessing cell interactions to decipher disease mechanisms. Lab website: https://www.medicalcellbiologylab.com/ Contact: https://www.researchgate.net/profile/Jeffrey-Aalders RE-place database: https://www.re-place.be/method/liquid-marbles-cost-effective-platform-generate-cardiospheres-co-cultured-cardiomyocytes-and
A hybrid in silico-in vitro cardiorespiratory simulator for medical device testing
Cardiovascular medical devices (CMDs) (e.g. artificial hearts, ventricular assist devices, ECMO, heart valves) support the cardiac and/or the respiratory function of patients. Large challenges are encountered when assessing CMDs interaction with the human body and the effects on the heart and vessels. Especially CMDs with new designs require an extensive evaluation concerning their effectiveness and safety under different pathophysiological conditions. We propose a high fidelity cardiorespiratory simulator for the testing of the hemodynamic performance of CMDs. The proposed simulator merges the flexibility of the in silico system with a hydraulic interface to test CMDs. As such, the simulator embeds a high fidelity cardiorespiratory model, allowing the reproduction of pathologies at both cardiac and respiratory level. The simulator works as a test bench for the assessment of CMDs, from prototype stage to pre-clinical stage. Thanks to its flexibility and high-fidelity, the simulator helps reducing animal testing and provides insights on how to improve CMD design to better suit different patient’s needs. Contact: https://www.kuleuven.be/wieiswie/en/person/00098489 RE-place database: https://www.re-place.be/method/cardiovascular-modelling-medical-device-testing
Large scale toxicoepigenetics on histones: a mass spectrometry-based screening assay applied to developmental toxicity
Toxicoepigenetics is an emerging field that studies the toxicological impact of compounds on protein expression through heritable, non-genetic mechanisms, such as histone post-translational modifications (hPTMs). Due to substantial progress in the large-scale study of hPTMs, integration into the field of toxicology is promising and offers the opportunity to gain novel insights into toxicological phenomena. Moreover, there is a growing demand for high-throughput human-based in vitro assays for toxicity testing, especially for developmental toxicity. Consequently, we developed a mass spectrometry-based proof-of-concept to assess a histone code screening assay capable of simultaneously detecting multiple hPTM-changes in human embryonic stem cells. To prove the applicability and performance, we first validated the untargeted workflow with valproic acid (VPA), a histone deacetylase inhibitor. These results demonstrate that our workflow is capable of mapping the hPTM-dynamics, with a general increase in acetylations as an internal control. To illustrate the scalability, a dose-response study was performed on a proof-of-concept library of ten compounds i) with a known effect on the hPTMs (BIX-01294, 3-Deazaneplanocin A, Trichostatin A, and VPA), ii) classified as highly embryotoxic by the European Centre for the Validation of Alternative Methods (ECVAM) (Methotrexate, and All-trans retinoic acid), iii) classified as non-embryotoxic by ECVAM (Penicillin G), and iv) compounds of abuse with presumed developmental toxicity (ethanol, caffeine, and nicotine). In conclusion, we show that toxicoepigenetic screening on histones is feasible and yields very rich data that can contribute to the (safe) development of drugs and holds potential, not only for applications in the pharmaceutical industry, but also for environmental toxicity and food safety. Lab website: https://www.progentomics.ugent.be/ Linkedin: https://www.linkedin.com/in/sigrid-verhelst-a54798172/ ResearchGate: https://www.researchgate.net/profile/Sigrid-Verhelst
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.
FirstbaseBIO - human brain organoids for studying neurological diseases
Human neurological diseases are still poorly understood, amongst others because animals are used as a model for the human brain. A way to overcome this problem is to mimic human brain functioning in a dish with organoids. FirstbaseBIO is developing off-the-shelf brain organoids on which neurological diseases can be studied. This 3D platform will be formed by reprogrammed human cells from easily accessible sources, for example urine, skin, or mucosa. The proof of-concept brain organoids will be those from patients who are suffering from adrenoleukodystrophy (ALD), a rare, incurable brain disease that occurs primarily in young boys and is often fatal. With the brain organoid platform, possible medicinal treatments for ALD can be effectively optimised. FirstbaseBIO was nominated for the Venture Challenge 2021 for their development of human brain organoids to study neurological diseases.
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/
Avatar Zoo - teaching animal anatomy using virtual reality
Animals are essential to train the next generation of scientists understand diseases and develop treatments for humans as well as animals. Therefore, animals are used for educational purposes. Technologies such as Virtual Reality and Augmented Reality can be employed to reduce the number of animals in the future. Prof. Dr. Daniela Salvatori is working on the development of 'Avatar Zoo' together with UMCU and IT. Live animals are replaced by holographic 3D in this flexible platform. With these holograms one is able to study the anatomical, physiological and pathological systems and processes of all kinds of animals. Avatar Zoo won the Venture Challenge 2021 for the development of virtual reality models that can be used for anatomy classes and practical training.