From a 3D-model of particle-induced granuloma-like structure to a simple macrophage bioassay predicting granulomagenic and fibrotic activity of particles
Scope of the method
The Method relates to
- Human health
The Method is situated in
- Basic Research
- Regulatory use - Routine production
Type of method
- In vitro - Ex vivo
This method makes use of
- Animal derived cells / tissues / organs
Description
Method keywords
- spheroids
- agarose
- ELISA
- cell culture
- confocal
Scientific area keywords
- lung toxicology
- predictive toxicology
- inhaled particles
- lung diseases
Method description
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 mimics granuloma formation and categorizes granuloma-inducing inorganic particles. Macrophage cell line (MHS) pre-exposed for 24h to 10µg/mL of granuloma-inducing (Carbon nanotubes, CNT) or not (Carbon black,CB) particles are cocultured 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 in presence of CNT but not CB were organized in layered compact cellular aggregates comparable to granulomas after 7 days. The supernatant collected at 24hours (but also at 78hours and 7days) contains significantly elevated levels of the pro-fibrotic mediator TIMP1 (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 combined to structural cells 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 were 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 new material and airborne dust particles.
Lab equipment
ELISA
cell culture infrastructure
(confocal) microscopy
Method status
- Still in development
- Internally validated
Pros, cons & Future potential
Advantages
- In vitro method
- no animal method : cell lines
- Simple
Challenges
Coating a flat agarose layer is necessary for observing a basal uniform cellular layer
Modifications
Refinement of the three cellular model to a unicellular model
Future & Other applications
Exposition to a wide range of reactive materials
References, associated documents and other information
Associated documents
CB (not-granuloma-inducing) - no spheroid.avi
vehicle - no spheroid.avi
CNT (granuloma-inducing) - granuloma-like spheroid.avi
From a 3D-model of particle-induced granuloma-like structure to a simple 2D-macrophage bioassay predicting granulomagenic and fibrotic activity of particles.pdf
Contact person
Léa HiéronimusOrganisations
Université Catholique de Louvain (UCL)louvain center for toxicology and applied pharmacology (LTAP)
Belgium