CSEM Scientific and Technical Report 2008

Online Detection of Cytokines for Monitoring Cellular Stress in Cell Cultures:
Application to Nanoparticle Toxicity Evaluation
S. Pasche, M. Giazzon, N. Matthey, G. Voirin
The need for assessing nanoparticle toxicity has become apparent with the increasing number of nanotechnology-based products on the market.
An analytical platform is being developed to evaluate nanoparticle toxicity in vitro, based on the detection of cytokines in cell cultures by means of
an optical biosensor.
Nanoparticles typically have a size of 100 nm and below. In
view of their high surface area, they have shown to lead to
increased biological activity and thus may exhibit toxicity,
often referred to as “nanotoxicity”. In recent years, a growing
number of products containing nanoparticles have appeared
on the market, for instance in cosmetics, food packaging,
sports equipment, paintings, glass windows, tiles, etc. In
addition, new opportunities in pharmacology and medicine
have been identified for drug delivery, medical diagnostics and
bio-imaging. The ongoing commercialization of nanotechnology
products will lead to increased human exposure to
nanoparticles. Consequently, evaluation of their potential
toxicity is essential. Whereas most reliable methods rely on
costly, in vivo experiments, in vitro assays present a promising
screening method for nanotoxicity assessments. To date, in
vitro cytotoxicity assays focus on cell viability, cellular stress
and inflammatory response.
In the European project DIPNA [1] , which aims at developing
an integrated platform for the in vitro investigation of
nanoparticle toxicity, CSEM is developing a tool for the
quantitative assessment of cellular stress caused by
nanoparticles. Here, the online monitoring of cellular stress in
cell cultures relies on the detection of cytokines that are
released by stressed cells into the medium. A label-free,
immunosensing, analytical device with optical detection allows
analysis of the cell culture medium for cytokine content in realtime
(Figure 1).
Figure 1: Integrated device for online detection in cell culture medium,
connecting the cell culture chamber to the fluidic cartridge of the
detection device.
Assessment of cellular stress relies on the detection of the
chemokine Interleukin-8 (IL-8) released by stressed cells into
the biological medium. A sandwich-type immunoassay with
the capture antibody immobilized on the surface and detection
antibodies in solution is used for quantifying IL-8 (Figure 1). A
photopolymerizable dextran based polymer coating
(OptoDex ® ) is used for the covalent immobilization of capture
antibodies on the surface and, at the same time, prevents non
specific interactions with the biological medium. Specific
binding of IL-8 and detection antibodies are monitored with the
wavelength interrogated optical sensing (WIOS) device which
uses the evanescent field of light to detect biomolecule
adsorption at the solid-liquid interface [2] .
60
IL-8 concentrations between 0 and 10 ng/ml within the cell
medium were detected with the implemented immunoassay
(Figure 2). The concentration of IL-8 released by human
bronchial epithelial cells (A549 cell line) exposed to an agent
that induces the production of IL-8 (induction factor rhTNF-α)
can be quantified. Comparison with reference measurements
(no exposure to the induction factor) allows discrimination
between naïve and stressed cells (induced with rhTNF-α) in
real-time (Figure 2, inset).
Figure 2: Real-time monitoring of the immunoassay for the detection
of various concentrations of IL-8 in cell culture medium, measured
with the WIOS instrument, showing the response signals related to
the binding of two consecutive detection antibodies. Inset: signal
response for the medium of naïve cells and cells induced by rhTNF-α.
Integration of an adapted fluidic cartridge is under
development and will allow implementing the detection system
in a broad platform for the continuous monitoring of cell
cultures. In addition, parallel detection of other relevant
cytokines will strengthen the analysis of the cell culture
medium, and provide an interesting alternative to current in
vitro methods for the analysis of nanoparticle toxicity on cells.
This development offers a new tool for direct analysis in cell
cultures. It will be applied to render “nanotoxicity” assessment
simple, fast and cost efficient. It is a first step towards a
screening method for nanoparticle toxicity, limiting the number
of costly in vivo experiments and impacting favourably on
occupational health safety.
This work is partly funded by the European Commission:
project FP6-NMP-STRP-032131. CSEM thanks them for their
support.
[1] www.dipna.eu
[2] www.dynetix.ch