W. Richard Bowen and Nidal Hilal 4

7.2 ENgINEERINg THE ECM FOR PRObINg CELL SENSINg 203
of parenchymal cells such as hepatocytes (liver cells) which require heterotypic
interactions between non-parenchymal cells (e.g. fibroblast) to
maintain their liver cell phenotype [30]. In this study, the microfabrication
approach allowed researchers to specify independent variables, including
the formation of a heterotypic interface and the ratio of cell populations
at specific locations in their samples, something which was not possible
using traditional random co-culture methods.
Although many of the above findings have been made possible with
the aid of micropatterning, they have also indicated the desirability of
further investigation at smaller length scales (�100 nm) where most of the
molecular mechanisms relevant to cell biology can be discovered. Thus,
we describe relatively recent investigations that have used nanopatterned
engineered surfaces in the next subsection.
Nanopatterning
To generate nanopatterns, electron beam lithography (EBL) is normally
used since the spatial resolution of photolithography is limited
by the diffraction of light. Rather than using a mask, EBL uses a focused
electron beam to directly write patterns onto an electron beam sensitive
resist. Since this is a serial writing method, i.e. tracks are written segment
by segment, this technique can require a significant amount of time to
write a single large area pattern and is thus very expensive. However, in a
development similar to the soft lithography described earlier, nanopattern
structures can also be imprinted onto a solid polymeric substrate (nanoimprinting
lithography [NIL]), greatly reducing the cost [31, 32]; Figure 7.4).
By combining NIL with self-assembled monolayer techniques, protein
nanopatterns of dimension �100 nm have been produced [33, 34]. The
combined capability of NIL and EBL for the generation of arbitrary nanopatterns
on a wide range of materials has also led to the discovery of cell
response to nanotopography, as discussed in later sections.
Other methods for nanopatterning biological molecules include scanning
probe lithography [35], self-assembly nanofabrication using block
copolymer, and colloidal lithography [36]. A good review of these techniques
is given by Gates et al. [37]. However, to generate a statistically
meaningful cellular study, fine tailored adhesive nanopatterns have to
cover a large area, preferably of the order of square centimetre. This
imposes a big challenge for some of the serial writing methods, such as
scan probe-associated lithography, although new developments in parallel
writing using multiple tips might mitigate this barrier.
As an example of an extension of the self-assembled block copolymer
technique, recently, Spatz’s group have developed the ‘micelle diblock
copolymer lithography’. This allows precise control of space between
RGD ligands at the length scale of 10–200 nm [38]. This strategy uses selfassembly
of diblock polymer of polystyrene-block-poly(2-vinylpyridine)