Biomedical Engineering Reference
In-Depth Information
By surface chemistry
By surface topography
>10 µm
PDL
Glass
PDL
PDMS
Glass
50-µm-deep step
PDL
PDMS
50 µm
FIGURE 6.40
In.vitro.guidance.of.axons.by.surface.chemistry.and.surface.microtopography..(From.
Li,.N..and.Folch,.A.,.“Integration.of.topographical.and.biochemical.cues.by.axons.during.growth.on.
microfabricated.3-D.substrates,”.
Exp. Cell Res.
.311,.307,.2005..Figure.contributed.by.Nianzhen.Li.)
he tip of the axon, also termed growth cone due to its morphology, is a highly specialized
cellular structure in charge of determining whether the substrate ahead is suitable for growth. In
time-lapse movies, the growth cone can be seen to extend inger-like protrusions (termed
ilo-
podia
) that seem to explore the substrate like blind “tentacles”; only if the “tentacle” adheres, the
axon grows ater it, extending more tentacles ahead. In vitro, if no soluble factors are used, axon
growth can also be microengineered by either modulating the surface composition (because the
axon follows the areas where the ilopodium is adhered) or the surface topography (because a
topographical change necessarily biases the exploratory range of the ilopodia), as depicted in
Figure 6.40
. hese studies not involving soluble gradients are technologically less challenging
and logically were developed irst, so they will be considered irst here.
6.5.1.1 Axon Guidance by Biochemical Surface Micropatterns
As early as 1987, Friedrich Bonhoefer and colleagues, then at the Max Planck Institute in
Tübingen, Germany, created an ingenious device for immobilizing membrane fragments
(
Figure 6.41
). he device contains microchannels molded in silicone rubber (in essence, PDMS)
from a photolithographically etched master. Atop the PDMS microchannels, a membrane (a
nucleopore ilter with pore size 0.1 μm) was placed, such that when a suspension of cell mem-
brane debris was lowed on top of the device and suction was applied through the microchan-
nels, the cell membrane debris were hydrodynamically trapped on the top surface of the ilter
until it gets clogged (an idea that has been successfully recycled recently by Shuichi Takayama's
laboratory to capture and pattern cells, see
Figure 2.42
in Section 2.6.2.6). Ater the ilter gets
clogged, and stripes of membrane are formed, the ilter is moved on top of another matrix that
has homogeneous porosity (not containing microchannels; not shown in
Figure 6.41
) and
another cell extract is added on top of the ilter; when suction is restarted, the membrane frag-
ments from this new cell extract get immobilized in the areas between the old stripes, forming
stripes of two diferent kinds. (Here, we note for the BioMEMS student that Bonhoefer and his
colleagues had just invented the irst microluidic microdevice in history—and the irst PDMS
device in history—6 years before George Whitesides published the irst microstamping article!)
Friedrich Bonhoefer's group devised this micropatterning technique so that they could test
the hypothesis that speciic molecules present in the membrane of cells from the optic tectum
were able to guide the growth of chick retinal axons. hey observed that temporal axons showed
a preference for growth on membranes of the anterior tectum (their natural target area) over
posterior tectum (
Figure 6.41b
), whereas nasal axons did not show a preference (
Figure 6.41c
).
