Evaluation and Development of the Calu-3 Cell Line for Nanoparticle Toxicity Studies

We have been using the Calu-3 cell line as a physiologically relevant in vitro model of the upper airways for drug delivery studies.  The same features which we find desirable from a drug delivery standpoint are also relevant to the evaluation of the toxicity of nanoparticles.  The Calu-3 cell line has been used extensively as a polarized drug transport model based on the formation of functionalized tight junctions when cultured under either liquid-interface or air-interface conditions. The additional advantage provided by this cell line is that the secretory function is retained, allowing this cell type to produce mucus on their apical surface when cultured under air-interface conditions.  The use of a cell monolayer allows for flexibility in the method of particle delivery and control over the delivered dose. 

Cellular toxicity will be measured through the use of a MTS (tetrazolium salt) assay to quantify metabolically active cells, NR (neutral red) assay to examine membrane permeability and lysosomal integrity, and a Live/Dead Fixable dye from Invitrogen to evaluate cell membrane integrity.  The MTS and NR assays are designed for 96 well plates so the seeding density, cell incubation time, amount of MTS and NR dyes, and dye incubation time were optimized for the 96 well plates.  This procedure currently requires the dosing of the Calu-3 cells in liquid-interface conditions prior to monolayer confluence.  To overcome this limitation, we are also using the Live/Dead Fixable dye with the Calu-3 cells grown to confluence under air-interface conditions on semi-permeable Transwell membranes.  To remove the mucus from the apical surface of the cell monolayer we investigated several mucolytic agents before settling on one which did not alter the polarized monolayer after washing as measured through transepidermal electrical resistance (TEER).  This allowed us to look at the role which the mucus plays in altering the toxicity of nanoparticles.  After exposure these monolayers were washed then imaged via confocal microscopy to evaluate the intact monolayer and flow cytometry for quantification of the detached cell monolayer.

For exposure experiments we have absorbed multiple glycoforms of the lipooligosaccharides (LOS) from non-typeable Haemophilus influenza (NTHi) and polyethylene glycol (PEG) to the surface of commercially available carboxylate modified polystyrene nanoparticles.  These particles were instilled onto the apical surface of the cells and incubated for up to 24 hours.  The cell viability after exposure to varying nanoparticles doses was evaluated using each of these methods.   

For the Calu-3 cell line the presence of the natural secretions on the apical surface reduced the toxicity observed in the Live/Dead stain when compared to particles directly applied to a washed surface of Calu-3 cell monolayer.  This suggests that for the Calu-3 cell line the MTS and NR assays in the 96 well plates overestimated the amount of cell toxicity for particles when compared to the more physiologically relevant confluent monolayer grown under air-interface culture conditions.