Microfluidic Devices for Quantifying the Role of Soluble Gradients in Early Angiogenesis - Mechanical and Chemical Signaling in Angiogenesis - page 47

Fig. 2 Schematic showing that cellular response to a chemotactic gradient depends not only on
gradient steepness but also on concentration magnitude, which is a function of the cellular
position along the concentration profile. There is an optimal concentration (center) for
chemotaxis at which signaling by motility factors (a function of concentration magnitude) and
directional bias of receptor clustering (a function of the gradient steepness) are sufficient to
induce chemotaxis. Clustered receptors (triangles) drive extensions from the cell membrane. At
concentrations below the activation threshold (left), signaling from bound receptors is insufficient
to induce migration. At concentrations above the saturation threshold (right), receptors are
saturated on all sides of the cell, resulting in no sustained directional bias in receptor binding
integrate and act on soluble gradients. Substrate independence also allows for
three-dimensional (3D) culture under controlled gradients, which is important
because native vasculature is inherently 3D. Third, reductive experiments on the
time scales, i.e., days or weeks, necessary to quantify the role of soluble gradients
in collective behaviors such as path-finding [ 13 ], inosculation [ 14 ], and maturation
[ 15 ] of nascent sprouts are only possible under stable concentration profiles. With
microfluidic devices that apply specified, substrate-independent, stable gradients,
we can quantitatively and multifactorially analyze how soluble gradients and other
inputs contribute to angiogenesis.
2 Background: Gradient-Generator Devices
for Cell Culture Studies
Previous devices to study soluble gradients, such as transwell (aka Dunn) assays [ 16 ]
and Zigmond chambers [ 17 ], are ill-suited to developing a quantitative, reductive
understanding of the microenvironmental regulation of angiogenesis. These devices
expose cells to transient gradients by permitting diffusion from a factor-rich to a
factor-empty chamber. The transwell assay, for example, separates the chambers
with a porous membrane; the Zigmond chamber separates them with a narrow
intermediate chamber. For these ''no-flow devices'' (Table 1 ), as concentrations in
the chambers equalize, cells are exposed to an attenuating gradient that may be
completely absent in only 2 h [ 18 ]. Chemotaxis is analyzed by determining the
fraction of cells that transit to the source. Because the gradient is transient, sprouting
morphogenesis, which takes several hours to days [ 13 ], cannot be assessed. Recent
improvements upon Zigmond chamber technology, such as the microslide device for
chemotaxis [ 19 ], can maintain a gradient for up to 2 days (Fig. 3 a). Likewise,
transwell assays have been modified to support 3D culture [ 20 ]. The time scale of the