Biomedical Engineering Reference
In-Depth Information
the University of Manchester (later, in 1990) 20 years ater Carter, when immunoluorescence
staining techniques for molecular imaging of cells were already in full swing. his group used
microfabricated 5-μm-thick copper stencil mask containing feature linewidths of approximately
20 μm to deposit cell-adhesive Pd islands onto substrates previously coated with cell-repellent
poly-2-hydroxyethyl methacrylate (poly-HEMA). he shape of the cells was conined to the
shape of the islands (
Figure 6.3a
and
b
). In their irst article, they demonstrated that cell pro-
liferation was a strong function of island size (
Figure 6.3c
). For Nil-8 ibroblasts, the DNA syn-
thesis (a more accurate measurement of cell proliferation) reached a cell proliferation minimum
below the “zero contact” line (reference point obtained from suspended cells) for small islands
and increased for larger islands (
Figure 6.3d
), a inding that was very similar on mouse embryo
cells. However, 3T3 ibroblasts did not display a DNA synthesis minimum (
Figure 6.3e
), which
suggested for the irst time that this cell type had a major requirement for contact.
In a follow-up article, in 1990, Ireland and colleagues produced micropatterns of various
shapes (circles, triangles, or lines). Using actin and vinculin immunoluorescence, they showed
that cytoskeletal organization in 3T3 ibroblasts depends on island shape. In particular, the focal
points tended to accumulate to the peripheries of circles, the apices of triangles, and the margins
of lines (most obvious when the island reached ~1000 μm
2
in size), whereas the total focal adhe-
sion area was independent of the shape of the cell. Cells tended to favor the formation of small
focal points, independently of island size—with the caveat that, given enough space, on large
islands, they would also form larger focal points; a good analogy, if we consider that the focal
points are the “feet of cells,” is that cells constrained to small islands are forced to tiptoe whereas
cells free to spread on large islands have space to both tiptoe and stomp on their feet. Cell
motility speed increased linearly with island size on linear islands. DNA synthesis (measured
a
b
Poly-HEMA
Poly-HEMA
Palladium
Palladium
50 µm
c
d
e
3T3K
NIL-8 24 hours
3T3K 48 hours
100
100
Unlimited contact
Unlimited contact
100 hours
5
80
80
Cell proliferation
as a function of
island size
4
60
60
3
40
40
DNA synthesis
in experiment 1 ( )
and 2 ( )
4 hours
Zero contact
2
DNA synthesis
in experiment 1 ( )
20
20
1
Theoretical
Zero contact
and 2 ( )
0
0
500
1000
Contact area µm
2
2000
4000
500
1000
Contact area µm
2
2000
4000
500
1000
Contact area µm
2
2000
4000
FIGURE 6.3
Effect. of. cell. shape. on. cell. proliferation.. (From. Charles. O'Neill,. Peter. Jordan,. and.
Grenham. Ireland,. “Evidence. for. two. distinct. mechanisms. of. anchorage. stimulation. in. freshly.
explanted.and.3T3.Swiss.mouse.ibroblasts,”.
Cell
.44,.489-496,.1986..Reprinted.with.permission.
from.Elsevier.)
