Biomedical Engineering Reference
In-Depth Information
Fig. 7 Quantitative
evaluation of NSC
enrichments in cultures with
different material surfaces.
The fraction of NSC
phenotypes in the cultures are
shown for (
open circles
)
nestin
+
b
III
-
or (
closed
circles
) both nestin
+
b
III
and
nestin
+
b
III
+
. Data are also
shown for neurosphere
cultures (
NS
). The averages
(
SEM) of five experiments
are shown. Reproduced from
Nakaji-Hirabayashi et al. [
85
]
with permission from
Elsevier, copyright 2007
a marker for differentiated neurons. The number of cells that expressed nestin and
b
III was expressed as the percent of the total number of cells in the culture flask
(Fig.
7
). Cells with a nestin
+
b
III
-
phenotype represented the most immature
population. Both nestin
+
b
III
-
cells and the total number of nestin
+
cells (nestin
+
b
III
-
plus nestin
+
b
III
+
) were most abundant on the surface with 10% COOH-thiol
(97
0.8%, respectively). Although the abundance of cells with
these phenotypes decreased with decreasing COOH-thiol content, approximately
85% of all cells exhibited the nestin
+
2.7% and 98
b
III
-
phenotype on the EGF-His-immobilized
surface with 0.01% COOH-thiol.
We also conducted experiments to compare our culture method with the standard
neurosphere culture. In the standard neurosphere culture, cell number increased
approximately nine times over 5 days. Immunostaining showed that the neurosphere
cultures contained 54
b
III
-
cells. This
demonstrated that the standard neurosphere culturing method was less efficient than
EGF-immobilized substrates for selectively expanding NSCs. Thus, the EGF-
immobilized substrates prepared from mixed SAMs with 10% COOH-thiol provided
the most efficient method for selective NSC expansion.
Cells that were expanded on EGF-immobilized substrates were harvested and
cultured on freshly prepared EGF-His-immobilized substrate for another 5 days.
These procedures were repeated up to four times. The rate of cell growth was not
affected by repeated subculturing; the average doubling time was 18.1
5.3% nestin
+
cells and 41
7.4% nestin
+
1.8 h. In
addition, immunocytochemical staining showed that, after four subcultures, cells
retained 93
2.0% nestin
+
populations. These results suggested that, in theory,
NSCs can be expanded by approximately 10
5
-to10
6
-fold within 20 days on the
EGF-His-immobilized substrates. Furthermore, these cells retained multipotency.
Under appropriate conditions, the cells differentiated to express
b
III, GFAP (astro-
cyte marker), and O4 (oligodendrocyte marker) with marked reductions in nestin
expression. The results of the differentiation assays suggested that expanded cells
