Endothelial Cell Invasion Assay
is defined as the sprouting of new
capillaries from preexisting blood vessels (Folkman
and Shing, 1992). The formation of new blood vessels
occurs in a variety of normal and pathologic conditions.
During embryogenesis or wound healing, neovascularization
is the result of a balance of stimulatory
and inhibitory angiogenic factors. Normally this
balance is strictly controlled. However, during the
development of many diseases, including inflammation,
retinopathies, and cancer metastasis, the angiogenesis
controlling mechanisms may fail and result
in formation of a pathologic capillary network
During vascular assembly, endothelial cells respond
to a variety of extracellular growth factors and different
molecules involved in cell-cell and cell-matrix
interactions (Gale and Yancopoulos, 1999). Signaling
molecules, more commonly associated with neuronal
development, also play an important role in capillary
formation during angiogenesis (Wang et al.
Soker et al.
The angiogenic response is considered to be composed
of a series of sequential steps, including degradation
of the basement membrane
endothelial cells, migration of the endothelial cells into
the surrounding tissues, proliferation and differentiation
of the endothelial cells, and finally formation of
new capillary vessels
(Folkman, 1986). Most of these
distinctive steps of the angiogenic process can be mimicked in vitro
. in vitro
assays classically rely on the
use of two sources of endothelial cells: large vessel
endothelial cells, such as bovine aortic endothelial cells
(BAEC), or human umbilical vein endothelial cells
(HUVEC) (Zetter, 1988; Springhorn et al.
, 1995), and endothelial cells of microvascular origin (Kråling et al.
1994; Lamszus et al.
, 1999). in vitro
assays are quantifiable
and may specifically address each step of the
angiogenic cascade such as basement membrane
disruption (metalloproteinase assays), cell migration
wounding; phagokinetic track assay; modified
Boyden chamber chemotaxis assay), proliferation
(thymidine incorporation assay), and tube formation
by endothelial cells in three-dimensional gels, such as
collagen or Matrigel
. Matrigel is a tumor basement
membrane matrix extract enriched with laminin.
During angiogenesis, the proliferating and migrating
endothelial cells are eventually organized into newly
formed capillary structures. Endothelial cells
origins are able to form tube-like structures in vitro
when growing in appropriate extracellular matrix
components. Matrigel considerably enhances tube formation
(Grant et al.
, 1985; Madri et al.
There are several aspects of cell culture assays
involving endothelial cells that need to be taken into
account for the accurate interpretation of the obtained
results. Prolonged cultivation of endothelial cells may
change their properties dramatically, including alterations
in activation state, karyotype, expression of cell
surface antigens, and growth properties. Another
problem is that all endothelial cells are not alike.
Microvascular endothelial cells differ in different
organs or even are different within blood vessels of the
same organ (Gumkowski et al.
, 1991). Different
endothelial cells produce different factors (cytokines,
growth factors, and their inhibitors) that make the
assay system even more complex for evaluation. From
that point of view, organ culture assay systems including
aortic ring assay (Nicosia and Ottinett, 1990) and
chick aortic arch assay (Muthukkaruppan et al.
are even more complex. In these assays, entire aortic
segments, including nonendothelial cells, are placed in Matrigel. In 1-2 weeks the segments are monitored
for the outgrowth of endothelial cells and for the
formation of three-dimensional tube-like structures.
The main drawback of this system is using adult aorta
endothelial cells in the case of the aortic ring assay or
microvascular endothelial cells obtained from embryonic
arch, which are composed of cells dividing
before exposure to angiogenic factors. Nevertheless
the ability of endothelial cells to form tube-like structures
in Matrigel makes this in vitro
cell culture model
the most faithful assay system.
This article describes the modification of a tube
forming assay in Matrigel in which a dense clump of
cultured endothelial cells is formed by cultivation of
the cells in a hanging drop of growth media. During
incubation, cells form dense "clump" at the bottom of
the drop. The sediment of the cells is transferred onto
a layer of Matrigel and is covered with a thin layer of
Matrigel in which an interconnecting network of
endothelial capillary tubes is formed rapidly. Tube formation
occurs through an ordered sequence of events.
Endothelial cells localized on the surface of the "cellular
island" first develop large, dynamic cellular protrusions
and then form small aggregates and cord-like
structures. These early cord structures are dense and
do not have lumens. Cells start to migrate to form a
complex network of tube-like structures. The advantages
of the assay are the ability to directly visualize
the changes in cell morphology and the ability of
endothelial cells to generate tube-like structures.
Endothelial cells in the "clump" can survive for more
than a few days, leaving adequate time for assessing
angiogenic reactions. Introduction of angiogenesisinducing
factors or cells into Matrigel at some distance
from the "island" allows one to test chemotactic
activity. The assay is readily quantifiable but utilizes
already mentioned problems characteristic of the in
vitro angiogenesis assay.
Another advantage is that in this assay, cultivated
endothelial cell lines can be used, which can give more
standard results than the use of primary cultures, or
II. MATERIALS AND
Growth medium: Dulbecco's modified Eagle's
medium (DMEM) with a high concentration of
glucose (Cat. No. 31966-021,GIBCO) supplemented
with fetal bovine serum (FBS, Cat. No. F 7524,
Sigma) at 10%, 100× penicillin/streptomycinx (Cat.
No. 15140-148, GIBCO).
-free Dulbecco's phosphatebuffered
saline (PBS, 2.7mM
, 137 mM
Trypsin-EDTA solution (Cat. No. 25200-056, GIBCO)
HEPES buffer solution 1M
(Cat. No. 15630-049, GIBCO)
Stock of Matrigel - basement membrane matrix (BD
Biosciences, Cat. No. 354234). Matrigel is supplied
frozen and is stored at -70°C.
tissue culture flasks, 48-well tissue culture
plates, Pasteur pipettes, 10-ml pipettes (sterile), and
syringe needles (23 gauge).
|FIGURE 1 Culturing cells in the hanging drops. (A)
the cell suspension in the middle of the inner side
of the lid. (B)
Turning over the lid with the hanging drops.
(C) Transferring the
cell "clump" with a syringe needle.
- For culture in 75-cm2 flasks, suspend murine
microvascular endothelial SVEC4-10 cells in culture
medium at a concentration of 2 × 105 cells/ml and plate
out 2-4ml of cell suspension/flask. Place the flasks in
a 37°C tissue culture incubator and incubate in an
atmosphere of 5% CO2 overnight.
- The next day, examine cells under an inverted
microscope fitted with phase-contrast objectives.
Choose the flasks with the cell density >80% of
- Remove media and wash the cells with 10ml
- Replace the PBS with 1 ml of trypsin-EDTA and
incubate at 37°C for approximately 1 min. Cell rounding
should be observed in the inverted microscope.
When the cells are rounded, detach them by strong agitation.
Add 10 ml of culture medium supplemented
with 10% FBS to the flask, pipette the cells up and
down five times, and transfer contents to a 15-ml centrifuge
- Centrifuge the cells at 800g for 5min at room
temperature. Resuspend the cells in 10ml DMEM/
FBS. Count an aliquot of the cell suspension with the
Coulter counter or in a hemocytometer.
- Centrifuge the cells at 800g for 5min at room
- Resuspend the cells in DMEM supplemented
with 20% FBS, 1M HEPES, pH 7.5, at a concentration
of 3 × 106 cells/ml.
- Dispense 1.0ml of DMEM/FBS into each well of
a 48-well tissue culture plate.
- Turn the lid of the plate upside down. Plate out
0.02 ml of the cell suspension in the middle of the inner
side of the lid. The cell suspension should form a drop
- Carefully turn over the lid and place it on the
plate. The drops of the cell suspension will hang over
the media in the wells (Fig. 1B).
- Incubate plates at 37°C in a tissue culture incubator.
During overnight incubation, cells form a dense
"clump" at the bottom of the drop.
- Defreeze the necessary amount of Matrigel
overnight at 4°C with rotation.
- The next day, examine all the wells under a
stereomicroscope and select those that have the most
compact, well-formed "clump" of cells.
- Place Matrigel on ice. Add 5 × DMEM and FBS
to obtain a final concentration of 1× DMEM and 10%
FBS. Keep the mixture on ice.
- Place a fresh 48-well plate on ice. Dispense
0.15ml/well of Matrigel/mix into the plate. Incubate
the plate at 37°C in an incubator for 3-4 hours. The
Matrigel should solidify.
- Carefully lift the lid of the plate with hanging
drops, stick the cell "clump" to the tip of a syringe
needle, and transfer the "clump" on the surface of the
solidified Matrigel in the 48-well tissue culture plate
- Cover the cell "clump" with 0.01 ml of Matrigel/
mix and incubate the plate for 15min at 37°C in an
- Add 0.3ml of DMEM/FBS in each well and
return the plate to the incubator. The cells will remain
viable for several days.
Using the protocol described in this assay, it is possible
to demonstrate the effects of compounds on the
ability of endothelial cell to form capillary-like tubes in vitro
. We have also used this protocol to cocultivate
endothelial cells with other cell types and to study the
influence of the molecules produced by these cells
on the ability of endothelial cells to form capillaries
|FIGURE 2 Mouse endothelial cells SVEC4-10 migrating from a dense "clump" of the cells into Matrigel
containing an angiogenic compound: (A) 0 h of cultivation, (B) 24h, (C) 48 h, and (D) chemotactic activity of
an angiogenic compound. E, endothelial cells; F, tumor cells.
- To obtain maximum viability of cells growing in the
hanging drops, avoid long exposure of the cells to
trypsin during harvesting cells from the 75-cm2 flasks.
- Choose only well-formed, round-shaped, dense
- When lifting the lid with the hanging drops, caution
must be exercised to avoid disrupting the cell
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