Biomedical Engineering Reference
In-Depth Information
We also found that a gold layer was not required nor advantageous for general use
of the technique.
First, the surface of a glass plate (60 mm
1 mm) was modified with
3-aminopropyltriethoxysilane (APTES) to introduce the amine functionality. The
surface amine was reacted with glutaraldehyde to introduce aldehyde, and then it
was exposed to a solution of NTA; NTA coupling occurred through the formation
of a Schiff base. Subsequently, the glass plate was immersed in a NiSO
4
solution to
chelate Ni
2+
ions. Finally, EGF-His was anchored to the Ni
2+
-chelated surface to
obtain an EGF-His-immobilized glass plate. The surface density of coordinated
EGF-His was 0.53
0.10
m
g/cm
2
, measured with the microBCA assay. The MIR-
IRAS spectrum of a cognate surface exhibited strong absorption at amide I
0
and
amide II
0
bands (the prime notation designates a deuterated condition), which
indicated the presence of EGF-His at the surface. The conformations of character-
istic secondary structures were determined from the amide I
0
band. The results
showed that the content of
b
-sheet and
b
-turn structures in surface-anchored EGF-
His was similar to those observed in native EGF in solution and in EGF-His
anchored to a Ni
2+
-bound surface created on an alkanethiol SAM.
To fabricate a culture module, we adhered a silicone frame to the edges of the
plate to enclose the sides; then, a polystyrene film was adhered to the top of the
frame to allow gas exchange. This culture module provided an area of 25 cm
2
and a
chamber volume of 2.5 mL. To test the modules, we injected dissociated rat
neurosphere-forming cells. After 4 days of culture, 5
60 mm
10
6
cells were obtained
per module. These cells could be subcultured in new modules. We performed six
passages by subculturing every 4 days. At each subculture, some of the recovered
cells were seeded to a freshly prepared module at a density of 3.0
10
4
cells/cm
2
.
The average number of cells harvested after each subculture was 5.4
10
6
cells per module. We determined that 95% of the total number of cells had a
phenotype of nestin
+
1.7
b
III
-
. In addition, the cells obtained after six passages could
be differentiated, under appropriate conditions, into three major lineages, including
neurons, astrocytes, and oligodendrocytes. This demonstrated that
the cells
maintained multipotency.
The culture module greatly facilitated homogeneous distribution of seeded cells
and cultivation of a large number of cells under identical conditions. In addition, the
module required a smaller volume of medium than standard cell culture systems.
Importantly, this modular system provides the great advantages of scalability and
safety because cell processing can be performed in a closed system. Thus, the
modules facilitate the production of cells that are safe for use in cell transplantation
therapies.
In a separate study [
88
], we synthesized EGF fused to a polystyrene-binding
peptide [
101
] (EGF-PSt) that could be immobilized on the surface of a tissue
culture polystyrene dish. This surface also permitted efficient expansion of NSCs.
Thus, EGF-PSt can be used to produce large quantities of pure NSCs in standard
laboratories.
