Biomedical Engineering Reference
In-Depth Information
filopodia extensions (Fig.
6
a), while shallower gradients of 2 ng/mL/mm were
ineffective (Fig.
6
b). As predicted, a higher number of filopodia were observed on
the cell edge facing higher VEGF concentrations. While the filopodia distribution
was found to depend on the gradient steepness, the total number of filopodia was
found to be a function of average VEGF concentration (Fig.
6
c). VEGF concen-
trations of 20 ng/mL and greater were observed to stimulate significant filopodia
extension (Fig.
6
). These data suggest a minimum VEGF activation threshold is
required to induce a migratory phenotype (*20 ng/mL) while a minimum VEGF
gradient steepness is required to induce directionality (*14 ng/mL/mm).
Although cell polarization and chemotaxis can be conveniently studied in 2D,
other important angiogenic processes such as sprouting morphogenesis and lumen
formation are inherently 3D. To reductively study how soluble concentration
profiles affect these processes, microfluidic devices that produce stable, quantita-
tively determined gradients within 3D matrices are required. In addition, the ideal
experimental platform would enable tuning of the soluble gradient independently
from choice of 3D matrix in order to probe the effects of matrix properties, such as
matrix biochemistry, density, or stiffness in a reductionist manner. Devices that
impose transient gradients or rely on flow through the cell culture chamber do not
meet this criterion, because choice of matrix will impact diffusivity of the soluble
cue, thereby altering the time-course of a transient gradient and/or the flow profile
through the matrix, making comparisons across matrices impossible.
To achieve stable, matrix-independent gradients, the microfluidic device
described previously can be slightly altered. To enable injection of matrix compo-
nents, the height of the culture chamber can be increased. To decrease the time
required to reach steady-state, the flow rates in the reagent channels is increased to
40 nL/min and the size of micro-capillaries is increased. In a 3D experimental study,
these device changes are not observed to increase convective flow within the culture
chamber because the matrix itself serves as a barrier to flow. To initiate 3D sprouting
morphogenesis, endothelial cells are cultured as a 2D monolayer on collagen-coated
beads. These cell-coated beads are then suspended within a fibronectin/collagen
matrix. Sprouting morphogenesis was quantified in matrices of identical fibronectin
concentration and varying collagen density from 0.3 to 2.7 mg/mL, resulting in
materials with storage moduli of 7-700 Pa [
69
]. While increasing the matrix density
increased the time required to reach a stable VEGF gradient, all matrices resulted in
quantitatively consistent gradients of 50 ng/mL/mm VEGF within 2 h, a time scale
that is much shorter than the experimental time scale of sprouting morphogenesis
(1-4 days). As matrix density increased, cellular behavior changed from being
mainly migratory to mainly proliferative (Fig.
7
). At a low density of 0.3 mg/mL,
isolated cells migrated into the matrix and underwent single cell chemotaxis. In
intermediate densities, cells coordinated their chemotaxis; cells migrated as multi-
ples at 0.7 mg/mL and as stable sprouts at 1.2 and 1.9 mg/mL. At a high density of
2.7 mg/mL, cell proliferated on the bead surface to form stumps that did not migrate
into the surrounding matrix. Together, these data demonstrate that matrix density
influences cell migration and proliferation, and stable sprouting morphogenesis
occurs only when these two cellular behaviors are in balance.
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