Biomedical Engineering Reference
In-Depth Information
a
Microjet arrays
Cell culture resevoir
c
Stage
I
II
III
IV
V
0
5
25
40
42.5
69
Time (min)
Sink manifold
Source manifold
C = 0
P = P
L
C = C
o
P = P
R
d
e
b
Open
reservoir
CXCL8
fMLF
Source
C = C
o
Sink
C = 0
100
50
100
50
1
0
0
C
C
o
—
-50
-100
-150-100 -50
-50
-100
0
50 100 150
-150-100 -50
0
50 100 150
50 µm
X (µm)
X (µm)
X (µm)
FIGURE 6.27
Chemotaxis.studies.using.a.microjet.gradient.generator..(From.Thomas.M..Keenan,.
Charles.W..Frevert,.Aileen.Wu,.Venus.Wong,.and.Albert.Folch,.“A.new.method.for.studying.gradient-.
induced. neutrophil. desensitization. based. on. an. open. microluidic.chamber,”.
Lab Chip
. 10,. 116-
122,.2010..Reproduced.with.permission.from.The.Royal.Society.of.Chemistry.)
then shiting it, then applying an inverse gradient of a diferent chemoattractant (
Figure 6.27c
).
Distinct responses to the two chemoattractants were recorded (
Figure 6.27d
and
e
). he unex-
pected presence of ''tethered cells'' and the fact that many cells responded to the second CXCL8
or fMLF gradients but not the irst CXCL8 gradient provides evidence that neutrophils have
initial activation states, and long-term chemical sensitivities that difer dramatically. he popu-
lation-based approaches and analyses provided by traditional methods are simply inefective at
parsing out how speciic complements of chemotactic factor receptors and their relative activa-
tion states inluence neutrophil migratory behavior.
hese devices require, at the very least, some amount of microluidics know-how (either
for fabrication or for modeling, or both). Why not produce a microluidic device that relies
on purely difusive processes (no low) to produce the gradient? In 2007, a team from Cornell
University led by Michael Shuler and Mingming Wu presented a three-channel gradient gen-
erator made in agarose (
Figure 3.88
in Section 3.9.5.2); agarose is readily available in biological
laboratories, it is inexpensive and safe to use, it molds well, and it is biocompatible (it has been
used in the past for chemotaxis in what is called the “under-agarose” assay). In Shuler and Wu's
device, the central channel contained the cells (similar to the microjets' central reservoir) and
the lateral channels acted as sink and reservoir for the chemoattractant (similar to the microjets'
sink and reservoir manifolds). HL-60 cells, a human promyelocytic leukemia cell line, were dif-
ferentiated into neutrophil-like cells by culturing them in 1.3% DMS culture media for 4 to 7
days, and attached to ibronectin-coated glass. Less than 5 minutes ater the addition of fMLP
(250 nM, gradient = 0.27 nM/μm) into the source channel, the cells started to move (
Figure
6.28
). Hydrogel devices are extremely useful when conducting long-term experiments because
nutrients and gases necessary for cell survival difuse readily through the hydrogel.
None of the designs shown above are capable of delivering very sharp chemical gradients:
the concentration ield goes from zero to maximum over a distance that is much larger than
the cell. How can a gradient be delivered in such a way that it is ensured that one end of the cell
“sees” zero concentration and the other end sees the maximum concentration—if possible for all
the cells? Mehmet Toner and colleagues at Harvard Medical School built a device that cleverly
uses the cells themselves as barriers for the chemotaxis agent (
Figure 6.29
). he device consists
of many channels, each of which has the width of a single neutrophil, so as the neutrophils
