90 M. C. W. Chen and K. C. Cheung
Microﬂuidic devices also give advantages in ﬂuid handling, mixing, separation,
5.1.1 Traditional Cell Culture Methods
In traditional, well-established cell culture techniques, Petri dishes, culture well
plates, and culture ﬂasks are used to maintain either a two-dimensional monolayer
of adherent cells below a layer of liquid culture medium, or a suspension of non-
adherent cells within the culture medium. These bulk methods may require cell
densities of hundreds of thousands to millions of cells per milliliter.
This two-dimensional petri dish-based method of cell culture has long been
used in molecular and cell biology. Recently, there has been increased interest
in three-dimensional cell culture, in which cells are embedded in a material that
mimics the extracellular matrix.
5.1.2 Two-dimensional Versus Three-dimensional Culture Methods
In traditional two-dimensional cultures, cells can be incubated until the monolayer
reaches conﬂuence, or maximum cell density. The culture environment can have a
dramatic inﬂuence on cell behavior, with different responses observed between
cells incubated in 2-D versus 3-D environments, such as modiﬁed morphol-
ogy, gene expression, and proliferation.
A large part of tissues is composed of
the extracellular matrix (ECM). The ECM comprises a network of proteins and
polysaccharides that are mainly excreted by ﬁbroblast cells. ECM proteins include
collagen, ﬁbronectin, elastin, and laminin. Other major components of ECM
include glycosaminoglycans. The ECM mechanically stabilizes the tissue, and the
ﬁbrous network gives both physical and biochemical signals to the cells (Fig. 5.1).
The ECM interacts with cells to inﬂuence their shape, function, and survival.
Mammalian cells communicate with each other through several mechanisms,
including cell-cell contact as well as cell-ECM interaction.
Cells are anchored to
the ECM through integrins, which are proteins on the cell surface. Integrins link
the cytoskeleton to the ECM. Speciﬁcally, ﬁbronectin is the key ECM protein for
cell attachment. The Arg-Gly-Asp (RGD) tripeptide sequence in ﬁbronectin has
been identiﬁed as the central component which binds to integrins on cell surfaces.
One major factor affecting cell phenotype, function, proliferation, and migra-
tion is cell shape.
Each cell type has a different context, or microenvironment
(Fig. 5.2). The 3-D environment is a major factor affecting the differentiation of
stem cells. Different cell signaling pathways are activated in 2-D and 3-D cell
culture, and these pathways are mediated in part by cell adhesion matrices.
D and 3-D adhesion sites differ in chemical composition and spatial organization,
with different integrin complexes formed depending on the culture geometry.
In cell-based drug screening, the different cellular responses exhibited in 2-D
versus 3-D culture will have a crucial impact in the pharmacological response to
drugs, which may differ between cells in 2-D and 3-D culture.
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