Biointerfaces Between Cells and Substrates in Three Dimensions - Biointerfaces: Where Material Meets Biology - page 145

6.3.3 Electron Microscopy
Electron microscopy is a powerful tool to probe cell structure and ultra-
structure and works perfectly well in three dimensions, again providing that
the substrate can survive the chemical processing procedure. Cells in three
dimensions can be imaged using both scanning electron microscopy (for cell
surface assessments) as well as transmission electron microscopy (for cell
ultrastructure assessments). The first step in the procedure is fixing the cells,
usually with a combination of paraformaldehyde and glutaraldehyde fixa-
tives followed by osmium tetroxide. For SEM experiments, samples are then
dehydrated through a series of ethanol gradients and then dried using a
critical point dryer. For TEM experiments, samples are embedded into a
hard resin after dehydration and then sectioned into ultra-thin sections
(typically 70 nm) before being stained and imaged.

d n 3 r 4 n g | 3

TIP: The 3D scaffolds intended for analysis via electron microscopy should be
chemically and mechanically stable. Scaffolds that are too soft and unstable
will not survive the extensive electron microscopy processing procedure; this
will likely destroy the 3D organisation of the cells before imaging.

Figure 6.9 shows a TEM image of a hepatocyte cell cultured on Alvetex s .
The morphology and ultrastructure of the hepatocyte is 3D, with healthy
signs of microvilli on the surface of the cell. The scaffold can also be seen in
the image.

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Figure 6.9 Ultrastructural analysis of HepG2 cells cultured on Alvetex s using
transmission electron microscopy. Sc, Alvetex s scaffold; N, nucleus;
Mv, microvilli.
Reinnervate is acknowledged for supplying the image and allowing
reprint in this chapter. Scale bar ¼ 2 mm.