(274a) 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.