Sbastien Cambier sebastien.cambier@list.lu The digestive track - PowerPoint PPT Presentation

INTESTINAL IN VITRO CULTURES AS A VALUABLE TOOL FOR HAZARD ASSESSMENT OF NANOMATERIALS Sébastien Cambier sebastien.cambier@list.lu

The digestive track Mouth + 1.5 min Chewing Oesophagus 2-6 hour Chyme Stomach production Nutrient Intestine 90 hours absorption 2

Key elements of the Intestinal Environment Esser et al., 2018 Joana Costa and Arti Ahluwalia, 2019

Intestinal in vitro model: Monoculture Bacteria, NPs, etc.  Standard model for intestinal barrier  Differentiation into enterocytes …However ... Brush border  More cell types present Caco-2/TC7, epithelial cells  Mucus present

Intestinal in vitro model: Co-culture Continuous mucus secretion Brush border  More cell types present  Mucus secretion

Intestinal in vitro model: Co-culture Presence Of The Mucous Layer : HT29-MTX % Mucus staining Structure observation 0 % 10 % 100 % Georgantzopoulou et al., PFT, 2016

Intestinal in vitro model: Co-culture TEER : Trans Epithelial Electric Resistance TEER Real epithelial barrier

Intestinal in vitro model: Co-culture Assessment of Ag NPs effects : Oxidative stress DCFH-DA Fluorescence Cell Wash and add NPs Add DCFH-DA measurement seeding DCFH-DA esterases DCFH +ROS Diffentiation & mucus development Incubation Exposure day 0 14 days 30 min 2 hours DCF Values are mean ± SD of 3 independent experiments

Intestinal in vitro model: Co-culture Assessment of Ag NPs effects : inflammatory response

Intestinal in vitro model: Co-culture Detection of Ag NPs : TEM Georgantzopoulou et al., PFT, 2016

Intestinal in vitro model: Co-culture Effects Ag NPs : Proteomics Hierarchical clustering PCA Spot Maps (Score Plot) 1.8 1.6 1.4 1.2 Ag 200 nm 1 0.8 0.6 0.4 Control 0.2 PC2 0 -0.2 AgNO 3 -0.4 -0.6 -0.8 -1 Ag 20 nm -1.2 -1.4 -1.6 -1.8 -2 -1 0 1 2 PC1 Georgantzopoulou et al., 2016

Intestinal in vitro model: Co-culture CONCLUSIONS In vitro assays should be physiologically relevant  Coculture to be closer to real condition  Mucus reduces the effects of exposure Ag NPs effects are not only caused by Ag-ions

 No OECD guideline for in vitro intestinal testing  No in vitro Digestion OECD guidelines  No OECD guidelines for Nanotoxicology

TG for in vitro toxicity LIST will have the main role

LIST

coordinators

CONCLUSIONS In vitro assays should be physiologically relevant  Coculture to be closer to real condition  Mucus reduces the effects of exposure  Harmonized by intra/inter laboratory round robin TG will become available for in vitro intestinal effects

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