Insights into the Role of Cell–Cell Junctions in Physiology and Disease - International Review of Cell and Molecular Biology - page 204

One main advantage of using cell sheet assays in contrast to cell aggregate
assays is that in the case of the former, the cells are plated normally until
immediately prior to the assay. While use of hanging drops or micropipette
aspiration is not without advantages, the fact that the cells remain in suspen-
sion for extended times may lead to changes in baseline behavior, which may
in turn affect adhesion readouts, for normally adherent cells. In the case of
micropipette aspiration, a clear readout of the actual adhesion forces is not
always easily obtainable, although there are mathematical models that can be
used to help provide some estimates. Use of micropipettes that flex can lead
to more direct extraction of forces using the beam equation, although plating
cells on the tips can be challenging. Alternative methods are being devel-
oped. For example, “teethed” plates that can be separated after cells attach
may eventually lead to force quantification ( Hui and Bhatia, 2007 ).
Another challenge is to determine the role of junctional proteins when
cell-cell contact is the dominant mechanism by which the cell receives
information regarding the environment. There appears to be some evidence
that cells need force generation to thrive; when the substrate is too soft, for
example, cells will tend to cluster and form cellular networks that they
otherwise would not on stiffer substrates ( Califano and Reinhart-King,
2008 ). Thus, it would appear that the machinery for cellular regulation
by junctional proteins is extant but is usually attenuated by cell-substrate
interactions. Indeed, the balance between cell-cell and cell-substrate forces
can be considered to be regulated together, perhaps by one unifying
mechanism ( Maruthamuthu et al., 2011 ). Consider that the matrix within
an organism is generally self-generated; thus, there may be promise in engi-
neering cells in the absence of external substrates ( Wei et al., 2011 ), since
cell-substrate contact is not necessary for cell survival ( Fig. 5.4 ).
Another approach to examine the properties of cell-cell interactions uses
a substrate coated with a cell-cell adhesion molecule and either a cell or
another object coated with a cell-cell adhesion molecule. Such studies have
the advantage that a particular protein (usually a receptor) is targeted but the
results are likewise limited to that single interaction. Such studies have been
done using atomic force microscopy and micropillars ( Ganz et al., 2006;
Rakshit et al., 2012 ).
5.2. Effects of junctional proteins on cell physiology
Despite the extensive molecular and clinical work on junctions, it is useful to
develop and apply in vitro methods for characterizing how junctional