research activities in 2007 - CSEM

Microfabricated Membranes for Cell Layer Culture and Analysis
T. Overstolz, A. Hoogerwerf, M. Liley, F. Spano
A microfabricated membrane chip is being developed for cell culture and analysis. Integrated Pt-electrodes allow the detection of intercellular
junctions and the evaluation of the ‘tightness” of epithelia cell layers. These tools are designed to screen the toxicity of nanoparticles, in particular
their capacity to cross biological barriers such as the lungs.
Nanomaterials based on nanoparticles have become very
popular for many applications, including extremely strong
composite materials based on carbon nanotubes. In parallel
with this development, there has been a growing interest to
study the toxicity of these nanoparticles to the human body. In
vitro methods for these tests are being developed in order to
complement and/or replace large scale animal screening
tests.
In the body, epithelial cells are organized in sheets of cells
that make up the epithelia. All epithelia have the function of
providing a barrier between the body and the external world.
In order to achieve this, individual epithelial cells are joined via
intercellular junctions that make the epithelium impermeable.
Thus, transport across the epithelia occurs essentially through
the epithelial cells (trans-cellular transport) rather than
between the cells (para-cellular transport).
In vitro models of epithelia must be tested for the presence of
intercellular junctions and the absence of gaps between cells,
to ensure that transport across the in vitro model closely
resembles that of the epithelium in vivo. One of the most
widely used approaches to determine the ‘tightness’ of a layer
of cells is to measure its electrical impedance. Electrical
impedances of around 200 Ohm/cm2 may be considered
representative of “good” model epithelia that may be used to
study transport processes.
In order to minimize the influence of artifacts that interfere with
impedance measurements in aqueous media (e.g.
electrolysis, electrochemical potentials, fouling of the
electrode surface), the impedance of cell layers is usually
measured using low frequency alternating currents in a
4-terminal sensing approach. In the 4-terminal sensing
method, one pair of electrodes is used to inject the current into
the system, while a second pair of electrodes measures the
potential across the cell layer.
Figure 1: Silicon chip with integrated platinum electrodes for
impedance measurement of intercellular junctions. The central cell
culture well with its yellow porous Si3N4 membrane is visible. The two
other square holes act as fluidics ports.
Figure 2: Close-up view of Figure 1 showing the square well with the
microporous Si3N4 membrane, inset shows a detailed view of the
hexagonal hole pattern.
To enhance the reproducibility of the measurements the
electrode distance and placement should be well defined.
CSEM has therefore opted to use photolithography to define
the electrodes, rather than the less precise shadow masking
Moreover, the precise electrode definition accommodates
2-terminal impedance measurements, which are still in use.
A microchip has been fabricated integrating a square well for
cell culture including a thin microporous silicon nitride
membrane, on-chip platinum electrodes, and inlets for a
microfluidic system to supply cell culture medium. A
photograph of the resulting chip is shown in Figure 1, whereas
a more detailed photomicrograph of the membrane is depicted
in Figure 2. Thin silicon nitride membranes are especially
suitable for this purpose since they are transparent and thus
compatible with the many optical analysis and detection
techniques used in cell biology.
The fabrication technology starts with the deposition and
structuring of the LPCVD nitride layer that will form the
membrane. The front side structuring defines the hole pattern
in the membrane and the backside defines the opening for the
subsequent etch of the substrate material. Prior to this etch,
when the wafers do not have much topography, platinum
electrodes are deposited and patterned using a photoresist
liftoff. Only after the electrodes are defined the substrate is
etched in KOH to form the membrane.
The next steps foresee the integration of the polymer-based
microfluidic system and the electrical measurements of the
cell layer tightness.
The project partners are the University of Glasgow and the
Katholieke Universiteit Leuven.
This work was partly funded by the European Commission
(Contract number 515843-2). CSEM thanks them for their
support.
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