Microfluidic Devices for Angiogenesis - Mechanical and Chemical Signaling in Angiogenesis - page 93

in vivo microenvironment. A common theme involves the culture of ECs in the
presence of different extracellular matrix components and angiogenic factors.
Although these models have been extremely valuable for understanding several
aspects of the cellular and molecular mechanisms operative in angiogenesis, they
lack the capability of including other important factors such as chemical gradients,
surface shear stress and interstitial flows, as discussed in more detail later.
Physiologically relevant and well-controlled models that better mimic normal
and pathological angiogenesis would narrow the gap between bench-top discov-
eries and clinical applications. In vivo models have physiological relevance yet
inherently lack a high level of control. On the other hand, in vitro models have the
potential for greater degree of control, yet lack critical elements of the in vivo
microenvironment. There remains much room for improvement, and this has
motivated many to explore microfluidic methods in the search for greater in vivo
relevance in an in vitro model.
1.2 Traditional Methods of Studying Endothelial Chemotaxis
Directional migration toward a gradient of soluble chemoattractant, or chemotaxis,
is an essential feature common to many cell types that is vital for organ devel-
opment, wound repair, inflammation, neurite outgrowth, angiogenesis, tumor
invasion and metastasis. During angiogenesis, activated endothelial cells degrade
their underlying basement membrane, interpret directional cues and migrate
toward a chemotactic gradient of angiogenic factors (e.g. VEGF, bFGF [ 11 ]).
A modified Boyden chamber assay, also known as the Transwell assay is widely
used [ 12 - 15 ] to study endothelial chemotaxis. The Boyden chamber consists of
upper and lower compartments separated by a porous filter barrier. Filters are
typically available in different membrane materials (e.g. polycarbonate, polyester,
and cellulose nitrate), surface treatments and pore sizes which are chosen based on
cells of interest. In this method, cells are seeded in the upper compartment and
chemoattractant placed in the lower. After a predetermined incubation period,
typically 4-6 h, the number of cells that have migrated across the entire width of
the filter is quantified. Two major weaknesses with this method include
(1) uncertainty in the nature of gradient generated and (2) the inability to visualize
and continuously monitor cells during the course of the experiment. Other tradi-
tional methods for studying endothelial migration include the wound assay [ 16 ],
Teflon fence assay [ 17 ] and phagokinetic track assay [ 18 ]. In each of these, there is
an inherent challenge in distinguishing between chemokinesis and chemotaxis.
Consequently, many in the research community have recently looked to micro-
fluidic approaches that facilitate the establishment of stable gradients and permit
real-time monitoring.