BIOLOGICAL CONSIDERATIONS

3.1 THE CULTURE ENVIRONMENT

The validity of the cultured cell as a model of physiological function in vivo has frequently been
criticized. Often, the cell does not express the correct in vivo phenotype because the cells
microenvironment has changed. Cellcell and cellmatrix interactions are reduced because the cells lack
the heterogeneity and three-dimensional architecture found in vivo, and many hormonal and nutritional
stimuli are absent. This creates an environment that favors the spreading, migration, and proliferation of
unspecialized progenitor cells, rather than the expression of differentiated functions. The influence of the
environment on the culture is expressed via four routes: (1) the nature of the substrate on or in which the
cells growsolid, as on plastic or other rigid matrix, semisolid, as in a gel such as collagen or agar, or
liquid, as in a suspension culture; (2) the degree of contact with other cells; (3) the physicochemical and
physiological constitution of the medium; (4) the constitution of the gas phase; and (5) the incubation
temperature. The provision of the appropriate environment, including substrate adhesion, nutrient and
hormone or growth factor concentration, and cell interaction, is fundamental to the expression of
specialized functions
3.2 CELL ADHESION
Most cells from solid tissues grow as adherent monolayer, and, unless they have transformed and become
anchorage independent after tissue disaggregation or subculture they will need to attach and spread out on
the substrate before they will start to proliferate. Originally, it was found that cells would attach to, and
spread on, glass that had a slight net negative charge. Subsequently, it was shown that cells would attach
to some plastics, such as polystyrene, if the plastic was appropriately treated with an electric ion discharge
or high-energy ionizing radiation. We now know that cell adhesion is mediated by specific cell surface
receptors for molecules in the extracellular matrix (see also Sections 8.4, 17.7.3), so it seems likely that
spreading may be preceded by the secretion of extracellular matrix proteins and proteoglycans by the
cells. The matrix adheres to the charged substrate, and the cells then bind to the matrix via specific
receptors. Hence, glass or plastic that has been conditioned by previous cell growth can often provide
a better surface for attachment, and substrates pretreated with matrix constituents, such as fibronectin or
collagen, or derivatives, such as gelatin, will help the more fastidious cells to attach and proliferate. With
fibroblast-like cells, the main requirement is for substrate attachment and spreading and the cells migrate
individually at low densities. Epithelial cells may also require cellcell adhesion for optimum survival
and growth and, consequently, they tend to grow in patches.



3.2.1 Cell Adhesion Molecules
Three major classes of transmembrane proteins have been shown to be involved in cellcell and cell
substrate adhesion. Cellcell adhesion molecules, CAMs (Ca2+ independent), and cadherins (Ca2+
dependent) are involved primarily in interactions between homologous cells. These proteins are self-
interactive; that is, homologous molecules in opposing cells interact with each other [Rosenman
& Gallatin, 1991; Alberts et al., 2002], and the cellcell recognition that this generates has a signaling
role in cell behavior [Cavallaro & Christofori, 2004]. Cellsubstrate interactions are mediated primarily
by integrins, receptors for matrix molecules such as fibronectin, entactin, laminin, and collagen, which
bind to them via a specific motif usually containing the arginineglycineaspartic acid (RGD) sequence
[Yamada & Geiger, 1997]. Each integrin comprises one and one subunit, the extracellular domains of
which are highly polymorphic, thus generating considerable diversity among the integrins. Both integrins
and cadherins interact with vinculin, a step in signaling to the nucleus [Bakolitsa et al., 2004]. The third
group of cell adhesion molecules is the transmembrane proteoglycans, also interacting with matrix
constituents such as other proteoglycans or collagen, but not via the RGD motif. Some transmembrane
and soluble proteoglycans also act as low-affinity growth factor receptors [Subramanian et al., 1997;
Yevdokimova & Freshney, 1997] and may stabilize, activate, and/or translocate the growth factor to the
high-affinity receptor, participating in its dimerization [Schlessinger et al., 1995].
Disaggregation of the tissue, or an attached monolayer culture, with protease will digest some of the
extracellular matrix and may even degrade some of the extracellular domains of transmembrane proteins,
allowing cells to become dissociated from each other. Epithelial cells are generally more resistant to
disaggregation, as they tend to have tighter junctional complexes (desmosomes, adherens junctions, and
tight junctions) holding them together, whereas mesenchymal cells, which are more dependent on matrix
interactions for intercellular bonding, are more easily dissociated. Endothelial cells may also express tight
junctions in culture, especially if left at confluence for prolonged periods on a preformed matrix, and can
be difficult to dissociate. In each case, the cells must resynthesize matrix proteins before they attach or
must be provided with a matrix-coated substrate.

3.2.2 Intercellular Junctions
Although some cell adhesion molecules are diffusely arranged in the plasma membrane, others are
organized into intercellular junctions. The role of the junctions varies between mechanical, such as the
desmosomes and adherens junctions, which hold epithelial cells together, tight junctions, which seal the
space between cells, e.g. between secretory cells in an acinus or duct or between endothelial cells in a
blood vessel, and gap junctions, which allow ions, nutrients, and small signaling molecules such as cyclic
adenosine monophosphate (cAMP) to pass between cells in contact Although desmosomes may be
distributed throughout the area of plasma membranes in contact they are often associated with tight and
adherens junctions at the apical end of lateral cell contacts As epithelial cells differentiate in confluent
cultures they can form an increasing number of desmosomes and, if some morphological organization
occurs, can form complete junctional complexes. This is one reason why epithelial cells, if left at
confluence for too long, can be difficult to disaggregate. As many of the adhesion molecules within these
junctions depend on Ca2+ ions, a chelating agent, such as EDTA, is often added to the trypsin during or
before disaggregation.

3.2.3 Extracellular Matrix
Intercellular spaces in tissues are filled with extracellular matrix (ECM), the constitution of which is
determined by the cell type, e.g., fibrocytes secrete type I collagen and fibronectin into the matrix,
whereas epithelial cells produce laminin. Where adjacent cell types are different, e.g., at the boundary of
the dermis (fibrocytes) and epidermis (epithelial keratinocytes), both cell types will contribute to the
composition of the ECM, often producing a basal lamina. The complexity of the ECM is a significant
component in the phenotypic expression of the cells attached to it, so a dynamic equilibrium exists in
which the cells attached to the ECM control its composition and, in turn, the composition of the ECM
regulates the cell phenotype [Kleinman et al., 2003; Zoubiane et al., 2003; Fata et al., 2004]. Hence a
proliferating, migratory fibroblast will require a different ECM from a differentiating epithelial cell or
neuron. Mostly, cultured cell lines are allowed to generate their own ECM, but primary culture and
propagation of some specialized cells, and the induction of their differentiation, may require exogenous
provision of ECM. ECM is comprised variously of collagen, laminin, fibronectin, hyaluronan,
proteoglycans, and bound growthfactors or cytokines . It can be prepared by mixing purified constituents,
such as collagen and fibronectin, by using cells to generate ECM and washing the producer cells off
before reseeding with the cells under study or by using a preformed matrix generated by the Engelberth-
Holm-Swarm (EHS) mouse sarcoma, available commercially as Matrigel Matrigel is often used to
encourage differentiation and morphogenesis in culture and frequently generates a latticelike network
with epithelial or endothelial cells. At least two components of interaction with the substrate may be
recognized: (1) adhesion, to allow the attachment and spreading that are necessary for cell proliferation
and (2) specific interactions, reminiscent of the interaction of an epithelial cell with basement membrane,
with other ECM constituents, or with adjacent tissue cells, and required for the expression of some
specialized functions and others explored the growth of cells on other natural substrates related to
basement membrane. Natural matrices and defined matrix macromolecules such as Matrigel, Natrigel,
collagen, laminin, and vitronectin (B-D Biosciences, Invitrogen) are now available for controlled studies
on matrix interaction. The use of ECM constituents can be highly beneficial in enhancing cell survival,
proliferation, or differentiation, but, unless recombinant molecules are used there is a significant risk of
the introduction of adventitious agents from the originating animal .
Fibronectin and laminin fragments are now available commercially
.
3.2.4 Cytoskeleton
Cell adhesion molecules are attached to elements of the cytoskeleton. The attachment of integrins to actin
microfilaments via linker proteins is associated with reciprocal signaling between the cell surface and the
Cadherins can also link to the actin cytoskeleton in adherens junctions, mediating changes in cell shape.
Desmosomes, which also employ cadherins, link to the intermediate filamentsin this case,
cytokeratinsvia an intracellular plaque, but it is not yet clear whether this linkage is a purely structural
feature or also has a signaling capacity. Intermediate filaments are specific to cell lineages and can be
used to characterize them The microtubules are the third component of the cytoskeleton; their role
appears to be related mainly to cell motility and intracellular trafficking of micro-organelles, such as the
mitochondria and the chromatids at cell division.