Assay and Drug Development Technologies - 23

3D APPROACHES
do offer a relatively high grade imaging surface and images can
be obtained from the very center of these wells, where the well
profile tends to promote the accumulation of3D structures.94,95
In many of these cases, similar to the hydrogels, 3D cultures
may need to be liberated from the plates and transferred to an
appropriate surface to facilitate analysis using a HCS. This
process adds to the labor intensiveness ofthe procedure as well
as the possibility of damaging the cultures.
Micropatterned surfaces. Other modified surface technology is
available in the form of micropatterns. The surface of these
plates is imprinted with microstructure patterning to control
cell adhesion and promote 3D cell culture. These plates are
specifically designed for 3D culture and have the advantage of
possessing a transparent bottom film to enable microscopic
observation, as well as availability in a selection of patterns
and adhesive properties to cater for a variety of cell types. It is
recommended that cells are optimized using all plate types to
discern the optimum culture conditions. Cells are seeded on
the plates; however, they cannot adhere strongly and migrate
randomly along the patterned surface. This migration encourages
the formation of cell-to-cell interactions and the
subsequent generation of 3D models in vitro. Cells use the
micropatterned surface as a scaffold which supports the 3D
models as they grow, and being loosely attached to the surface,
they can be harvested easily by simply removing with a
pipette.96 These plates have many features, which make them
compliant with HTS. For example, micropatterned plates have
little well-to-well and plate-to-plate variation; therefore it
should be possible to generate 3D models of uniform size
using this technology. Optimization utilizing the different
patterns and adhesion properties infers control over 3D model
location and geometry. The plates come ready to use in, so far,
as they do not require any other treatment or extra equipment.
Caution is advised, however, as bubbles can easily form in the
media, which may disturb the culture. Damage can also occur
to the micropatterned surface itself if care is not exercised
when pipetting. One must also be careful when refreshing the
culture medium.96 While many micropatterned surfaces are
compliant with HCS, as with many anchorage-independent
technologies, their use is restricted only to the microplate
formats currently available.
Hanging drop. Hanging drop technology is a well-known 3D
cell culture system. HDPs are available in both 96- and 384well
formats and are composed of an assembly stack, which
allows culture of cells, media exchange, and harvesting.
Hanging drops are created by carefully pipetting up to 50mLof
cell suspension into wells from the top of the plate. Plate geFig.
4. Diagram illustrating a HDP with cell suspensions. HDP,
hanging drop plate.
ometry allows the drops to form and suspend from the bottom
of the wells through surface tension. The cells are confined
within this small droplet and are thus encouraged to interact
and aggregate (Fig. 4).
Three-dimensional cell culture using this method allows the
user to exert control over the size of the cultures generated,
aiding reproducibility and promoting compatibility with many
HTS instruments. To image the 3D models produced by the
hanging dropmethod, theymust be harvested and transferred to
an appropriate plate. This can be achieved either by centrifugation
or by the addition of excess media to the hanging drops.
These transfermethods require careful optimization and caution
as they both put the integrity ofthe cultures at risk. Care must be
taken to reduce evaporation of the hanging drops; hence some
plates are equipped with peripheral reservoirs designed to buffer
against and combat this issue. Care must be taken to not contaminate
the wells with the chosen buffer solution. Even though
this method of3Dmodel formation is generally compatible with
HTS, a significant level oftechnical expertisemay be required to
gain maximum impact from these products.97-99 While the
standard HDP systems are in a 96-well plate format, 384-well
HDPs have recently become available at similar pricing to the
96-well products and therefore represent a significant saving
per assay.100 However, in our experience, 96-well HDP systems
are complex in terms ofliquid handling, and the recent advance
to 384-well format may be beyond the liquid handling capability
of some laboratories.
Collodial suspension medium. After examining and trialing a
number of technologies available on the market, we sought to
engineer our own. We aimed to design a one-step system that is
capable of accommodating cells from initial seeding, through
3D model formation to final analysis in the same culture vessel,
while also being compatible with HTS and HCS. We felt that an
anchorage-independent CSM would best achieve this. Our optimized
cell suspension medium is of low viscosity; a feature
which has been uniquely formulated to comply with liquid
handling systems, HTS, HCS, and flow cytometry. It allows cells
the freedom to naturallymigrate, selfaggregate, proliferate, and
ª 2022 MARY ANN LIEBERT, INC. ASSAY and Drug Development Technologies 23
Assay and Drug Development Technologies

Table of Contents for the Digital Edition of Assay and Drug Development Technologies
