3.2.3 Three-Dimensional Basement Membrane Assay
The 3D ECM plays an important role as a critical framework and cytokine
immobilization scaffold for EC migration, connection, lumen formation
and tube stabilization(Senger and Davis, 2011).By thickening the
basement membrane or by adding an additional layer, the previously
described 2D basement membrane assay can be easily converted into a 3D
scaffold model(Deroanne et al., 1996).The 3D scaffold’s bottom, middle,
or top can be covered with single cells or EC monolayers. On a
fibronectin, laminin, or gelatin-coated dish, or at the surface of a
collagen gel, ECs attach, spread, and multiply to form a monolayer. If
the collagen layer is removed after covering with a collagen layer as a
3D matrix, ECs gradually organise into a network of tube-like
structures, which then regress(Deroanne et al., 1996).Collagen type I
and fibrin matrix appear to be particularly conductive to EC tube
morphogenesis and sprouting(Davis and Senger, 2005). Fibroblasts
cultured in the same conditions do not form a network, indicating that
the 3D basement membrane is important for network formation as the
specific behavior of ECs(Deroanne et al., 1996).When ECs are mixed into
the 3D matrix, most of them participate in the morphogenic response. In
contrast, in the case of the EC monolayer, only a subset of ECs
participates in sprouting formation(Senger and Davis, 2011). The
formation of an EC lumen in the 3D matrix has been observed and found to
be dependent on the formation and coalescence of pinocytic intracellular
vacuoles(Bayless and Davis, 2003). Compared to the 2D test, the 3D
basement membrane inspection offers several advantages and is now one of
the most used models for quantitative 3D angiogenesis in
vitro(Bahramsoltani et al., 2009). It allows ECs to build structures
that are not only capillaries, but lumens also. The movement of ECs in
both horizontal and vertical aspects can be detected and analysed
readily(Bayless and Davis, 2003).Fluidic flow is also applicable to
understand the effect of shear stress on EC behaviour (Kang et al.,
2008). However, the 3D basement membrane assay has limitations. These
assays, for example, take significantly longer to run (5-15 days)(Staton
et al., 2009).Furthermore, the 3D matrix thickness must be relatively
small to allow oxygen and nutrients to diffuse while avoiding excessive
cell mortality
4. Apoptosis assay (Annexin V-FITC)
This assay is performed by seeding the appropriate cell type and
treatment with the test compounds (in serum free media) in their
IC50 concentration for the predetermined time-points at
5% CO2 and 37 ◦C in a humidified
atmosphere. Untreated cells will be used as a negative control.
Post-treatment with the test compounds, cells are harvested by
trypsinization followed by PBS washing to clear off the cell debris.
This step is followed by the addition of Annexin V-FITC, mixing and
incubation in dark. The cells are then resuspended in binding buffer and
incubated with propidium iodide (PI). The cells are analysed by flow
cytometry(Kntayya et al., 2018).
5. Brine Shrimp Lethality Bioassay
The brine shrimp lethality assays are used to measure the cytotoxicity
of the test compounds(Naher et al., 2019). In this experiment, brine
shrimp eggs (Artemia salina) were incubated for 48 hours in a tank
filled with sterile artificial seawater at 28–300 C
with adequate aeration (using an air pump) and continuous light (60 W
lamp). Following the collection of nauplii using a Pasteur pipette,
brine shrimp are inserted into each well containing seawater. The serial
dilution technique is used to create test solutions at various
concentrations in individual vials filled with 5 mL of seawater
containing 10 nauplii shrimps. After each vial has been examined under a
microscope for 24 hours, the number of nauplii that survived is counted.
The information is used to calculate the nauplii%, mortality of brine
shrimp for both the control and increasing concentrations, and
LC50 is calculated. The reference standard is potassium
dichromate.(Lung and Cells, 2020).
Cell viability assays & cytotoxicity study
The different types of cytotoxic and cell viability assay include (a)
dye exclusion assay such as Trypan blue, erythrosine B
assay, (b) colorimetric assay such as MTT assay, MTS
assay, LDH assay, SRB assay, NRU assay & crystal violet assay,
(c)fluorometric assay such as Alamar Blue assay and
CFDA-AM assay (d) luminometric assay such as ATP assay
& real-time feasibility assay.