Biointerfaces Between Cells and Substrates in Three Dimensions - Biointerfaces: Where Material Meets Biology - page 141

The main disadvantage of the material is surface chemistry. Whilst
polystyrene is the conventional substrate for in vitro cell growth, it is not
an optimum surface for cell growth. To rectify this, Alvetex s can be easily
coated with ECM proteins such as collagen or fibronectin to improve cell
adhesion and migration. Furthermore, recent studies have also shown that
the material can be chemically functionalised with ECM mimics, such as
pendent galactose residues, to enhance cell adhesion. 15

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6.3 Methods to Examine Cell Behaviour in Three
Dimensions: Alvetex s Case Study
Materials that offer a 3D interface for cell growth must still allow researchers
to obtain information about the biological status of the cells during and after
the growth period. There is little point culturing in three dimensions if a
researcher cannot 'talk' or 'listen' to cells in that environment. Being able to
image cells, examine their cell structure, assess gene expression and
understand protein activity are just some of the crucial needs of researchers
working in the field of 3D cell culture, regenerative medicine and tissue
engineering. The remainder of this chapter will therefore focus on some
examples of examining cell behaviour in three dimensions, using the poly-
meric Alvetex s scaffold as an example to illustrate basic methods. It is im-
portant to note that these methods are merely intended to highlight a few of
the general procedures applicable for examining cells at the 3D biointerface,
and that more detailed protocols of each technique can be obtained from the
Reinnervate website (http://reinnervate.com/).

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6.3.1 Imaging of Live Cells
Constantly monitoring cell status under a bright-field light microscope is
standard practice for 2D cultures; however, this is much more dicult at the
3D interface. Three-dimensional cultures approximate the complexity and
structure of native tissue, creating issues with light scattering and thus
preventing clear, crisp images from being obtained. Furthermore, most 3D
substrates are not transparent, which again limits the use of conventional
bright-field microscopes for direct imaging. In order to image live cells in
three dimensions, either the use of a chemical stain or an advanced confocal
microscope is needed.
For Alvetex s , it was found that applying a common non-toxic stain
(neutral red; Sigma) was a very useful way of gaining an indication of cell
attachment and confluency with a standard bright field microscope. The
stain, which only needs to be exposed to the cells for a matter of minutes,
quickly penetrates through cell membranes and accumulates in the lyso-
somes. Imaging with a standard bright field microscope then offers a simple
red visualisation of the cells (Figure 6.6). The stain can also be subsequently
washed away, allowing the culture to proceed as normal, although it is