Biomedical Engineering Reference
In-Depth Information
that expressed nestin retained, for the most part, multipotency and could differenti-
ate into neuronal and glial lineages.
Based on those results, we concluded that, when cultured on the EGF-
His-immobilized surface prepared from a mixed SAM of 10% COOH-thiol, highly
enriched NSC populations could be produced in large quantities. Over a 5-day
culture on the substrate, cells were expanded 32 times. These expanded cells
consisted of 98% nestin
+
cells that retained multipotency for differentiation into
neuronal and glial lineages. This suggested that selective expansion could be
repeated for large-scale production of highly enriched NSC cells.
3.4 Structural Integrity and Stability of Immobilized EGF-His
We wondered why NSCs proliferated exclusively on surfaces with EGF-His ligands
anchored by coordination. We focused on two aspects in particular: the conformational
integrity of coordinated EGF-His and the stability of coordinate bonds at the interface.
Conformational information was acquired with multiple internal reflection-infrared
absorption spectroscopy (MIR-IRAS) [
97
]. The stability of coordinate bonds was
assessed by culturing NSCs on a surface with a small region of EGF-His ligands
anchored by coordination. This spatially restricted EGF-His anchoring enabled an
intuitive exploration of EGF-His release under cell culture conditions.
The results of these studies showed that EGF-His coordinated to the Ni
2+
-
chelated NTA-surface retained an intact conformation and was firmly anchored to
the surface during NSC culturing procedures. Both attributes are essential for
establishing adherent cultures and, hence, selective expansion of NSCs. In contrast,
with covalent immobilization, the structural integrity of EGF-His was reduced due
to multivalent linkages to the surface. In physical adsorption, EGF-His maintained
its intact conformation, but was readily released from the surface during cell
culturing procedures. We concluded that structural integrity and firm anchorage
were optimized with the coordination method.
3.5 Spontaneous Dimerization of EGF
Numerous studies have shown that EGF binding to EGFR triggers receptor dimer-
ization. This is considered a crucial step in intracellular signal transduction [
98
].
Inspired by this mechanism, we designed EGF chimeric proteins that spontaneously
dimerized (dEGF-His). These dimers were terminally anchored to the substrate.
We expected that these preformed dimeric EGF structures would facilitate the
formation of EGF-EGFR dimer complexes more efficiently than monomeric
EGF structures.
