Biomedical Engineering Reference
In-Depth Information
Membrane A stripes forming
a
Membrane A suspension
Low pressure channel
Capillary pore filter
Silicone matrix
Porous glass frit
Plastic support
0.09 mm
7 mm
b
c
Temporal axons
Nasal axons
Day 2
100 µm
FIGURE 6.41
Hydrodynamical.immobilization.of.membrane.fragments:.the.biochemical.stripe.assay.
for.axon.guidance..(From.Jochen.Walter,.Brigitte.Kern-Veits,.Julita.Huf,.Bernd.Stolze,.and.Friedrich.
Bonhoeffer,.“Recognition.of.position-speciic.properties.of.tectal.cell.membranes.by.retinal.axons.
in
vitro
,”.
Development
.101,.685-696,.1987..Figure.contributed.by.Friedrich.Bonhoeffer.)
his setup, which became known as the “
stripe assay
,” was used by neurobiologists for many
years for the limited use of patterning axons, without realizing the technique had an immense
potential for patterning other biological solutions, including cells, and building a rich variety of
microluidic architectures.
Uwe Drescher (who had trained with Bonhoefer), also at the Max Planck Institute at
Tübingen, Germany, was interested in elucidating why only temporal axons respond to repel-
lent axon guidance cues of the caudal tectum in the stripe assay. He irst changed the cell culture
support surface (the ilter), which was somewhat impractical for microscopy, and changed it to
a solid support (plastic surface) coated with nitrocellulose. his coating procedure traces back
to seminal work in 1987 by Vance Lemmon and Carl Lagenaur (University of Pittsburgh), who
reasoned that “since nitrocellulose is a convenient substrate for rapid noncovalent attachment
of proteins, we coated sterile Petri plates with nitrocellulose dissolved in methanol.” Drescher's
group also used a soluble chimeric ephrinA2 molecule in which the hydrophobic C terminus
is replaced by the Fc part of a human immunoglobulin (ephrinA2-Fc), making it insensitive
to cleavage by PI-PLC. Drescher observed that overexpression of ephrinA ligands on temporal
axons abolished the sensitivity previously observed by Bonhoefer's group (see
Figure 6.41
),
whereas treatment with PI-PLC both removed ephrinA ligands from retinal axons and induced
a striped outgrowth of formerly insensitive nasal axons (
Figure 6.42
). his work suggests that
diferential ligand expression on retinal ephrinA5 axons is a strong determinant of topographic
targeting in the projection of retinotectal axons.
Philip Hockberger and colleagues, then at Bell Labs, pioneered in 1998 the use of surface
chemistry to microengineer neuronal cell attachment and axon growth. In their case, the sur-
face patterns were produced by photolithography, using aminosilane SAMs as the adhesive areas
