test facilities

Complex aerosol cell culture exposure

CERTAM and INSERM U644 are partners since 1996 on complex atmospheres biological impact thematic. Throughout many years of research and development, several biological material exposure systems have been set up and a cell culture diffusive exposure model has been developped during MAAPHRI european project (Multidisciplinary Approach to Airborne Pollutant Health Related Issues).

Complex aerosol diffusive mechanisms exposure system

The aerosol is leaded trough the exposure chamber from the central entrance and diffuses over the culture plates.

Adhesive cells are grown on micro-porous media allowing a direct exposure on the cell apical pole, cells are feeded trough the basal pole.

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Using several exposure chambers simultaneously and using different cell strains allows to set up many conditions experiments or increase the experiment number:

• Many atmosphere dilutions in parallel
• Extended cell culture area for monocellular studies
• Many replicates or many cell strains simultaneous studies

Cell culture laboratory.

CERTAM has a small laboratory allowing most of cell culture operations.

Equipment:

• Vertical laminar flow hood
• Cell culture incubator
• Centrifugation
• Warm bath
• Liquid nitrogen for freezing operation

Cell strains commonly used

• pGL3-transformed cells with different interleukin gene promotors
• Lung alveolar epithelial cells: A549 strain
• Lymphoblastic T cells: Jurkat strain

Complex atmosphere exposure organotypic culture model

Llung slices organotypic culture exposure model has been developed trough the partnership between CERTAM and INSERM.

This model preserves the alveolar structure as well as the inter-cellular interaction from peripheral lung.

Lung slices preparation

• Lung slices are obtained with a Vitron slicer apparatus and are 150µm thick and 8mm diameter.
• Biological samples are exposed 3 hours to diluted engine emissions into specially equipped incubators.

Biological evaluation

• Oxidative stress evaluation: glutathione assay, Superoxide dismutase, Catalase, Glutathione peroxidase.
• DNA damages evaluation: free nucleosomes assays, caspases, DNA ladders.
• Cell viability evaluation: ATP/ADP assay, LDH leaks.
• Inflammatory response evaluation: Pro and anti-inflammatory cytokines ELISA immunoassays.
• Immunohistochemistry: transmembrane protein staining (ICAM-1), TUNEL staining, 8OH-guanosine staining.
• Gas phase free radicals assay with ESR (Electronic Spin Resonance).

Publications

• Development of a new in vitro system for continuous in vitro exposure of lung tissue to complex atmospheres: Application to diesel exhaust toxicology
• Toxicity of diesel engine exhausts in an in vitro model of lung slices in biphasic organotypic culture: induction of a proinflammatory and apoptotic response
• Biphasic culture of rat lung slices for pharmacotoxicological evaluation of complex atmospheres

Contact: david.preterre@certam-rouen.com