Biomedical Engineering Reference
In-Depth Information
To overcome these limitations, we have developed culture substrates that enable
the highly efficient expansion of specific cells in adherent cultures [
37
,
85
-
88
]. An
important characteristic of these substrates is that specifically engineered growth
factors are immobilized on the surface. Extensive protein engineering techniques
were used to optimize the presentation of growth factors to cells.
3.1 Strategy for Adherent Cultures of Neural Stem Cells
NSCs, capable of self-renewal and differentiation into multiple cell types, are found
in embryonic and adult tissues of the central nervous system (CNS) [
89
]. Several
studies have demonstrated that NSCs are a potential source for cell replacement
therapies in CNS disorders [
90
,
91
]. Those studies have largely relied on the ability
to culture NSCs in vitro. To develop culture substrates for the selective expansion
of NSCs, we first considered the responsiveness of NSCs to growth factors.
Originally, NSCs were discovered as EGF-responsive cells from rodent CNS tissue
[
11
]. Another study [
36
] showed that the expression of EGFR on rat neurosphere-
forming cells was highly correlated to the expression of nestin, an intracellular
marker for NSCs. In addition, EGF was shown to be a mitogen for NSCs. Based on
these results, we hypothesized that a substrate with surface-immobilized EGF might
selectively trap NSCs from a heterogeneous population of cells. Furthermore, the
EGF-EGFR interactions that would specifically capture NSCs might also promote
NSC proliferation due to EGFR signaling. To test this hypothesis, we focused on
surface immobilization of EGF for the selective expansion of rat NSCs.
3.2 Oriented Immobilization of Engineered EGF
There are many protein immobilization techniques available. One of the standard
techniques uses amine chemistry, where surface-bound nucleophilic groups react
with amines, which are abundant in proteins [
92
]. Although this technique provides
covalent immobilization of proteins, the use of amines would cause protein
inactivation. In addition, it does not provide control over the orientation of the
immobilized protein to ensure efficient recognition by ligands. On the other hand,
recombinant DNA technology can overcome these problems. A recombinant pro-
tein can be designed that has a specific peptide motif at a given site in the molecule
for surface anchoring.
We used recombinant DNA technology to design unique substrates for in vitro
expansion of rat NSCs. For example, we fused a small peptide sequence of six
consecutive histidine (His) residues to the C-terminus of human EGF (EGF-His).
This EGF-His was anchored to the surface of a glass-based substrate by coordina-
tion with Ni
2+
ions, which were fixed on the surface of a SAM of alkanethiol.
Strikingly, neither a covalently immobilized EGF-His nor a physically adsorbed
