Biointerfaces Between Cells and Substrates in Three Dimensions - Biointerfaces: Where Material Meets Biology - page 143

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Figure 6.7 Live 3D imaging of dividing CHO-K1 cells on Alvetex s . Cells were stained
with CellTracker t CM-DiI (1/1000 dilution) immediately prior to seeding
onto the material. After 5 days, cultures were imaged on a ZEISS LSM 510
confocal microscope. Cells were stained with Hoechst 33342 the evening
prior to imaging. White arrows illustrate the dividing cells.
Reinnervate is acknowledged for supplying the image and allowing
reprint in this chapter. Scale bars ¼ 20 mm.

TIP: For 3D imaging using fluorescent markers and confocal microscopes, it
is crucial that the fluorescent marker employed produces a good signal-
to-noise ratio. This often requires extensive optimisation of the marker-
staining process. A poor signal-to-noise ratio masks positive fluorescent
staining and thus gives low-resolution images of the cells during confocal
microscopy.

There are some potential drawbacks to be aware of for such live imaging
techniques. First, operating confocal microscopes for live 3D imaging re-
quires specialist training, is time-consuming and needs the appropriate
equipment (e.g., stage inserts are required to maintain 37 1C and 5% CO 2 for
most animal cell experiments). Second, repeated and/or long-term exposure
to dyes and fluorescent light can sometimes lead to photobleaching and/or
light toxicity, jeopardising experimental results. Under certain working
conditions there may be the issue of auto-fluorescence by the 3D substrate
although this can be reduced by optimisation of the technique. Nonetheless,
the tool is highly powerful and probably the most applicable way of visual-
ising live cells at the 3D interface. An example of live cell imaging on
Alvetex s is shown in Figure 6.7, using CellTracker t CM-Dil and Hoechst
counter-staining to monitor dividing cells in three dimensions.

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6.3.2 Histology and Immunocytochemistry
Histological and immunocytochemical analysis of tissue samples is a well-
known method of analysing cell structure and status at a particular time
point. Hospitals frequently use these techniques to assess biopsy tissue
samples from patients as a tool to diagnose certain diseases, including
cancers. These techniques can easily be applied to cell cultures at the 3D
interface, providing that the 3D substrate is suciently permeable and
chemically stable to withstand the processing procedures.