Biomedical Engineering Reference
In-Depth Information
As described earlier, the surface density of immobilized EGF-His was shown to
be directly correlated to the COOH-thiol content. Therefore, we reasoned that the
initial cell attachment must involve an interaction between immobilized EGF-His
and an EGFR expressed on the cell membrane. In fact, reverse transcriptase
polymerase chain reaction assays revealed that neurosphere cells expressed EGFR
mRNA. In addition, cell adhesion to immobilized EGF-His was totally inhibited
when soluble EGF was added to the culture medium at 20 ng/mL. Flow cytometry
analyses further demonstrated that rat neurosphere cultures could be significantly
enriched in NSCs by plating cells onto a glass surface with immobilized EGF-His.
All these findings supported our hypothesis that cell adhesion was mediated by an
EGF-EGFR interaction.
Table
1
summarizes the results of proliferation assays carried out on EGF-
His-immobilized surfaces. The total cell number after 5 days of culture varied
with different surfaces. These differences could be attributed principally to
differences in the numbers of cells initially attached, demonstrated by the cell
numbers observed at 24 h. The doubling time, determined for the 24-72 h period
after cell seeding, was similar for all the surfaces, with an average of 16.9
4.7 h.
The mitogenic signal of EGF might have been saturated on all surfaces, including
the surface with the lowest EGF density. The doubling time determined on the
EGF-His-immobilized surfaces was approximately half the doubling time deter-
mined for the neurosphere culture (31.2
1.4 h). On the surfaces with 0.01, 0.1,
and 1% COOH-thiol content, the growth rate declined slightly after 96 h; this was
probably due to a reduction of EGF activity after 4-5 days.
NSC content was assessed in a population of cells expanded on EGF-
His-immobilized surfaces for 5 days. Cells were immunocytochemically stained
with antibodies specific for nestin, a marker for NSCs, and
b
-tubulin III (
b
III),
Table 1 Proliferation of neurosphere-forming cells in cultures with or without EGF-immobilized
surfaces
COOH-thiol
content
a
Surface density
of EGF-His
b
Cell number after
24 h
c
Cell number after
5 days
c
Doubling
time
d
(ng/cm
2
)
(10
4
cells/cm
2
)
(10
4
cells/cm
2
)
(%)
(h)
0
51
0.7
0.3
11.7
5.1
n.d.
0.01
76
1.2
0.4
38.6
17.6
16.8
5.5
0.1
110
1.4
0.2
46.4
17.2
16.7
5.2
1
215
1.6
0.1
49.6
15.2
17.2
5.4
10
342
1.8
0.2
56.7
10.5
17.3
5.9
100
390
2.5
0.2
64.0
6.6
17.0
4.0
1.4
e
Reproduced from Nakaji-Hirabayashi et al. [
85
] with permission from Elsevier, copyright 2007
a
Content in the solution used to prepare the mixed SAMs
b
Determined with a microBCA assay
c
Mean
Neurosphere
-
-
-
31.2
5 assays
d
Determined from the logarithmic plot of growth curves for the culture period of 24-72 h. Mean
standard deviation for
n
¼
4 assays
e
Determined for cells during the standard neurosphere culture
standard deviation for
n
¼
