Our series of Educational Webinars aims to promote the use and development of New Approach Methodologies by putting the existing knowledge in the spotlight!

Practical information

The Webinars are open to all interested parties including scientists, regulators, the authorities, and the public. All webinars will be recorded and will be placed on the RE-Place YouTube Channel. Participation is free of charge, but registration is mandatory.

Second session

The second session will take place on the 14^th of September from 4 to 5 pm CET via Webex and will zoom in on the development and optimisation of cellular models.

First, Léa Hiéronimus of the UCL will present his work titled 'From a 3D-model of particle-induced granuloma-like structure to a simple 2D-macrophage bioassay predicting granulomagenic and fibrotic activity of inhaled particles'. After obtaining a Master’s degree in Biomedical Sciences at the UCLouvain (Belgium), Léa Hiéronimus (he/they) is currently a PhD student in the LTAP laboratory (Louvain’s center for Toxicology and Pharmacology). The subject of his thesis is on the hazardous effects of inhaled inorganic pollutants, specifically on the formation of the lung granulomatous fibrosis. To help industries predicting which materials will induce this pathology if inhaled, Léa has taken the challenge of developing new simple bioassays relying on cell lines.

This presentation will be followed by Amar van Laar (UGent) who will discuss his work on the ‘Metabolism and Health Effects of Rare Sugars in a CACO-2/HepG2 Coculture Model’. Amar obtained Biomedical and Nutrition Science degrees in the Netherlands, before starting his PhD at Ghent University. He has contributed to several molecular research projects on nutrition and health, mostly focusing on metabolic health and cancer. Within these projects, he has also worked on developing innovative in vitro models, which could be interesting from both a research and animal welfare perspective.

Abstracts

• From a 3D-model of particle-induced granuloma-like structure to a simple 2D-macrophage bioassay predicting granulomagenic and fibrotic activity of inhaled particles – Lea Hieronimus

Macrophages orchestrate reactive particle segregation, compact aggregates of immune cells and non-immune cells and promote fibrosis-surrounding granulomas.

We developed a simple 3D in-vitro model that mimic granuloma formation in vivo and categorize granuloma-inducing particles. Macrophage cell line (MHS) pre-exposed for 24h to 10µg/mL to granuloma-inducing (Carbon nanotubes, CNT) or not (Carbon black, CB) are co-cultured with fibroblasts and epithelial cells (respectively MLG and LA4 cell lines) on 0,3% agarose coated wells.

Fluorescent dyes and confocal microscopy showed that these cells were organized in layered compact cellular aggregates comparable to granulomas after 7 days in presence of CNT but not CB. The supernatant collected at 24hours (but also 78hours and 7days) contains significantly elevated levels of the pro-fibrotic mediator TIMP-1 (metallopeptidase inhibitor 1) only in granuloma-inducing conditions (CNT). The levels of other pro-granulomagenic and fibrotic mediators (such as matrix metalloproteinase 1, MMP-1; Osteopontin, OPN or the chemokine CCL2) were not increased. Our data suggest that macrophages respond to granuloma-inducing particles by releasing TIMP-1 and organizing in vitro granuloma-like spheroids.

This model was further simplified, as MHS macrophages alone are sufficient for the specific release of TIMP-1 in response to granulomagenic particles. Quantification of macrophage-produced TIMP-1 is a novel and simple tool for predicting and assessing granuloma-inducing inorganic materials.

• Metabolism and Health Effects of Rare Sugars in a CACO-2/HepG2 Coculture Model - Dr. Amar Van Laar

Introduction: In vitro models can be used for a multitude of research purposes with the advantages of providing high-throughput options, having a relatively low cost and not harming living organisms. This combination makes in vitro models ideal for molecular research. However, many of these models are often considered as not sufficiently predictive for the real life context. As a result, studies with in vitro models are often overlooked by policymakers, many journals demand to confirm the results with in vivo models and they are often not perceived as a valuable alternative for animal models. We at NutriFOODChem have worked on making in vitro cell models more realistic and aim to increase the knowledge of nutrition and health through these models.

Method: Innovative co-culture procedures were used to allow interactions between different cell types, which simulates the interorgan interactions of the in vivo context. In this case, intestinal Caco-2 and hepatic HepG2 cells were used to study the impact of rare sugars (as new generation of sugar replacers) on liver health, epigenetics and non-alcoholic fatty liver disease (as published in “Nutrients”). Caco-2 cells were seeded on transwell inserts, formed the upper layer and were differentiated before sugar exposure. HepG2 cells were seeded on the bottom plate and had 24-hour interaction with the Caco-2 cells prior to the exposure.

Results: The Caco-2/HepG2 co-culture was suitable to obtain information on the impact of rare sugars on liver health. Sugars altered hepatic gene expression and affected both the cellular energy metabolism and hepatic fat accumulation. Rare sugars had a smaller impact on the energy metabolism and hepatic fat accumulation (compared to maltose and glucose), although individual differences were observed. The largest differences on the epigenetic level were observed between L-arabinose and glucose.

Discussion: The findings suggest that a specific subset of rare sugars holds a potential as sugar replacers. Related to the model, the study indicates that co-culture models could be used to increase the relevance of cell research within the metabolic health context. The Caco-2/HepG2 co-culture incorporated the in vivo aspect of intestine/liver interaction, and allowed to study the effects of disaccharides (following digestion and intestinal absorption) on the liver. Although this co-culture model cannot capture the entire complexity of the in vivo situation and may not yet replace in vivo models, it shows again that engineering of in vitro cell models can help to bridge the gap. These co-culture models may already be the preferred option to screen the effects of a large number of compounds. Furthermore, developing the concept further (eg towards organs-on-a-chip), may eventually reduce the dependency on animal models.

More information & other sessions

Information on the upcoming sessions will be published soon. In the meantime, don’t hesitate to contact us via info@-replace.be if you have any questions.

Sources:

• RE-Place YouTube channel: https://www.youtube.com/@re-place1330
• From a 3D-model of particle-induced granuloma-like structure to a simple macrophage bioassay predicting granulomagenic and fibrotic activity of particles: https://www.re-place.be/method/3d-model-particle-induced-granuloma-structure-simple-macrophage-bioassay-predicting
• Metabolism and Health Effects of Rare Sugars in a CACO-2/HepG2 Coculture Model: https://www.re-place.be/index.php/method/vitro-coculture