Biomedical Engineering Reference
In-Depth Information
In 2004, Dennis Discher's group from the University of Pennsylvania tested the hypoth-
esis that muscle cells sense both their molecular and mechanical microenvironment by cul-
turing them on collagen strips attached to glass or polymer gels of varied elasticity (
Figure
6.12
). Although fusion into myotubes occurred independently of substrate lexibility, myosin/
actin striations formed only on gels with stifness typical of normal muscle (Young's modulus,
E ~
12 kPa). Cells did not form striations on glass and much soter or stifer gels. Also, myotubes
grown on top of a compliant bottom layer of glass-attached myotubes (but not soter ibroblasts)
formed striations (
Figure 6.12a
and
b
), whereas the bottom cells only assembled stress ibers
and vinculin-rich adhesions (
Figure 6.12c
). As time progressed, striation of the top (but not
the bottom) myotubes increased (
Figure 6.12d
and
e
). Unlike sarcomere formation, adhesion
strength increased monotonically versus substrate stifness with strongest adhesion on glass.
To mimic neuromuscular synaptogenesis processes in vitro, the author's laboratory at the
University of Washington in Seattle devised a microluidic platform capable of sustaining long-
term perfusion and focal stimulation of an array of single C2C12 myotubes (
Figure 6.13
). he
device is assembled atop a pattern of lines of dried Matrigel separated by cell-repellent PEG-
IPN (
Figure 6.13a
; see also
Figure 2.29
in Section 2.6.1.2). C2C12 myoblasts are then injected
into the device, allowed to attach, and the nonattached cells are removed, leaving myoblast line
patterns within the microluidic device (
Figure 6.13b
). he cells are perfused in the direction
orthogonal to the main channel (parallel to the lines) for approximately 1 week in low-serum
conditions (so they do not divide) until they fuse into myotubes. At this point, clusters of ace-
tylcholine receptors (AChRs) form spontaneously on the myotube surface and the myotubes are
E
C2C2
= 12-15 kPa
a
Rigid glass
b
c
Upper myotube
Lower myotube
Actin
Myosin
Merge
10 µm
d
e
100
Upper myotube
Actin
Myosin
80
Upper myotube
60
Merge
40
20
10 µm
Lower myotube
0
1
2
3
4
Time (weeks)
FIGURE 6.12
Micropatterned.myotubes.provide.optimal.stiffness.for.myotube.striation..(From.Adam.
J..Engler,.Maureen.A..Grifin,.Shamik.Sen,.Carsten.G..Bönnemann,.H..Lee.Sweeney,.and.Dennis.
E..Discher,.“Myotubes.differentiate.optimally.on.substrates.with.tissue-like.stiffness:.Pathological.
implications.for.soft.or.stiff.microenvironments,”.
J. Cell Biol.
.166,.877-887,.2004..Figure.contrib-
uted.by.Dennis.Discher.)
