SLIDE 1 INTESTINAL IN VITRO CULTURES AS A VALUABLE TOOL FOR HAZARD ASSESSMENT OF NANOMATERIALS Sébastien Cambier
sebastien.cambier@list.lu
SLIDE 2 2
The digestive track
Mouth + Oesophagus Stomach Intestine 1.5 min 2-6 hour 90 hours Chewing Chyme production Nutrient absorption
SLIDE 3 Key elements of the Intestinal Environment
Esser et al., 2018 Joana Costa and Arti Ahluwalia, 2019
SLIDE 4
- Standard model for intestinal barrier
- Differentiation into enterocytes
Caco-2/TC7, epithelial cells Brush border Bacteria, NPs, etc.
- More cell types present
- Mucus present
…However...
Intestinal in vitro model: Monoculture
SLIDE 5 Continuous mucus secretion Brush border
- More cell types present
- Mucus secretion
Intestinal in vitro model: Co-culture
SLIDE 6 Presence Of The Mucous Layer : HT29-MTX %
Georgantzopoulou et al., PFT, 2016
Mucus staining Structure observation
0 % 10 % 100 %
Intestinal in vitro model: Co-culture
SLIDE 7
TEER : Trans Epithelial Electric Resistance TEER Real epithelial barrier Intestinal in vitro model: Co-culture
SLIDE 8 Diffentiation & mucus development Incubation 30 min 14 days
Cell seeding Add DCFH-DA
Exposure
Fluorescence measurement
2 hours
Wash and add NPs
day 0
Values are mean ± SD of 3 independent experiments DCFH-DA DCF DCFH-DA DCFH +ROS esterases
Intestinal in vitro model: Co-culture
Assessment of Ag NPs effects : Oxidative stress
SLIDE 9
Intestinal in vitro model: Co-culture
Assessment of Ag NPs effects : inflammatory response
SLIDE 10 Georgantzopoulou et al., PFT, 2016
Intestinal in vitro model: Co-culture
Detection of Ag NPs : TEM
SLIDE 11 PC2 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2
- 0.2
- 0.4
- 0.6
- 0.8
- 1
- 1.2
- 1.4
- 1.6
- 1.8
PC1 2 1
Spot Maps (Score Plot)
Control Ag 200 nm Ag 20 nm AgNO3
PCA Hierarchical clustering
Georgantzopoulou et al., 2016
Intestinal in vitro model: Co-culture
Effects Ag NPs : Proteomics
SLIDE 12
In vitro assays should be physiologically relevant Coculture to be closer to real condition Mucus reduces the effects of exposure
Intestinal in vitro model: Co-culture CONCLUSIONS
Ag NPs effects are not only caused by Ag-ions
SLIDE 13
No OECD guideline for in vitro intestinal testing No in vitro Digestion OECD guidelines No OECD guidelines for Nanotoxicology
SLIDE 14
TG for in vitro toxicity
LIST will have the main role
SLIDE 15
LIST
SLIDE 17
TG will become available for in vitro intestinal effects
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
SLIDE 18
Merci villmols
