Assay and Drug Development Technologies - 22

RYAN ET AL.
the well surface may render some hydrogels noncompliant with
HCS imaging systems. To culture 3D models in a hydrogel and
analyze them using a HCS, 3D structures may need to be recovered
from the matrix and transferred to a fresh vessel. As well
as adding extra steps to an already relatively labor intensive
workflow, if not handled with care transferring 3D models between
vessels could potentially compromise their integrity.87
Inert matrices. Another anchorage-dependent method of
generating 3D cell culture models is the utilization of inert
matrices. An inert matrix is composed of a rigid, porous
scaffold and engineered into thin membranes that fit into
conventional cell culture plastic ware, ranging from Petri
dishes to 384-well plates.88 The aim of these matrices is to
provide cells with the space and freedom to proliferate, interact
and differentiate as they would in vivo. The inert nature
of the scaffold removes the issue of contamination arising
from animal sources, while cells are never further than 100 mm
from the nutrient medium owing to a membrane thickness of
200 mm and a pore size of 36-40 mm. While originally designed
for generic 3D culture of cells, specific applications
have now been developed for these scaffolds. These include
in vitro cosmetic and drug testing as well as toxicity and cell
invasion assays.77 As efficient as this system is at culturing
cells in 3D format, it is quite labor intensive. The plates may
require rehydrating and wash steps before cell seeding and, in
a similar manner to hydrogels, the structure of the scaffold
itself makes this system unsuitable for HCS. In fact, cells
contained in this matrix cannot be viewed under a light microscope
without the addition of a visible dye. In our laboratory,
we have also witnessed auto-fluorescence in these
plates. Certain lipophilic dyes, Nile red, for example, also
have a tendency to bind to the scaffold, which may further
hinder imaging. Recovering the cultures generated is generally
not an option. To do so, they would require treating
with trypsin followed by agitation,89 both of which could
potentially compromise their integrity. Analysis is required
in situ, generally by absorbance assays, such as MTT [3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide] and
MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)2-(4-sulfophenyl)-2H-tetrazolium].
These scaffolds have been
utilized in conjunction with a variety of cell types, including
various cancer lines such as HepG2 (human hepatic carcinoma)90
and HT-29 (human colorectal adenocarcinoma),91 and
have successfully produced 3D models suitable for downstream
processing. However, the labor intensive factor associated
with this technology, with regard to setup, imaging,
and cell recovery, cannot be overlooked. While inert scaffolds
may be suitable for small scale experimentation, there are
22 ASSAY and Drug Development Technologies
technologies available which are more compliant with HCS
systems as discussed in later sections.
Anchorage-Independent Technologies
Anchorage-independent (scaffold-free) 3D culture methods
promote self aggregation of cells in the medium by providing
more freedom to move as opposed to the spatial restrictions
imposed by scaffolds. Opting to go scaffold free means a
product will be selected from one of the following categories:
low adhesion plates; micropatterned surfaces; or hanging
drop plates (HDPs).
Low adhesion plates. Low adhesion plates consist of a polystyrene
surface often treated with hydrophobic or hydrophilic
coatings, which renders them biologically inert therefore
greatly reduces the binding of attachment proteins or cells.
This encourages the natural migration of cells toward each
other and the formation ofan ECM.92 A drawback of these 3D
culture systems is the issue of reproducibility. Low adhesion
plates possess the capability to produce large numbers of 3D
models. However, we have discovered in the course of our
work that to generate cultures of a uniform size, much optimization
with regard to cell seeding density is required and
even after optimization uniformity is not guaranteed.
Low adhesion surface technology is applied to a range of
tissue culture vessels, including both 96- and 384-well plates.
In addition to the standard flat bottom version, these plates are
also available in the form of U-bottom and V-bottom wells,
the latter promoting miniaturization by requiring less culture
medium. This feature encourages the movement of cells toward
each other and promotes the formation of 3D models
in culture medium, potentially in a shorter period oftime than
if left to aggregate naturally.93 By utilizing this method, the
incidence of generating single cultures of uniform size in the
troughs of these wells is increased, which is ultimately compatible
with HTS. In both examples, the vessels arrive precoated,
which reduces the labor involved, therefore all that is
required by the end user is to seed their cells as they see fit.
The potential production of reproducible 3D models in a
short space of time using these methods make them ideal
candidates for HTS technologies.93 They are designed in such a
way as to reduce cross talk and background noise in luminescence
and some fluorescence assays,78 howevertheydonot
lend themselves well to HCS. High-content imaging requires
the use of high image grade quality plates to obtain sufficient
image resolution to perform the downstream image analysis.
Treatments on the low adhesion plates can interfere with the
imaging surface and the plates are required to be flat bottomed
to capture the highest quality images. Certain U-bottom plates
ª 2022 MARY ANN LIEBERT, INC.

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