Biointerfaces Between Cells and Substrates in Three Dimensions - Biointerfaces: Where Material Meets Biology - page 142

d n 3 r 4 n g | 3

Figure 6.6 Neutral red staining of CHO-K1 cells cultured in Alvetex s for 2 days and
imaged using a standard bright-field microscope. Note the clear visual-
isation of the cells even though light is scattered around the 3D interface.
Reinnervate is acknowledged for supplying the image and allowing
reprint in this chapter. Scale bar ¼ 200 mm.

.

generally recommended to use sacrificial cultures where possible as any
residual stain may impact the experiment.

TIP: For 3D imaging with neutral red staining, dye exposure will likely
need to be optimised for cell type, scaffold and the microscope being used.
However, a useful starting point is the neutral red protocol for Alvetex s ,
described on the Reinnervate website (http://reinnervate.com/using-alvetex/
protocols/).

Using confocal microscopy in conjunction with a fluorescent marker
(such as CellTracker t CM-Dil; Invitrogen) is another tool to visualise live
cells in three dimensions. With this method, optical sections are used to
construct impressive 3D depth profiles (Z-stacks) of the entire cell mass in
real-time. This allows for important structural changes to be monitored
during cell differentiation, division or migration in three dimensions. Fur-
thermore, depending on the thickness of the sample and the capacity of the
microscope, almost entire 3D tissue blocks (typically 100 mm) can be
examined, offering useful insights into the organisation of cells at the 3D
interface.