Page 186 - Veterinary Toxicology, Basic and Clinical Principles, 3rd Edition

Toxicological Testing: In Vivo and In Vitro Models Chapter | 9 153

VetBooks.ir more closely in models of developmental toxicity albeit at (Hansen and Inselman, 2011). These cell lines are nor-
mally maintained in growth media containing mitogens,
a much higher financial cost compared to the well-
and can be induced to differentiate into different cell
established tumor cell lines, such as those mentioned
earlier. types (e.g., cardiomyocytes, pancreatic, and neural cells)
Clonal cell lines are derived from the mitotic division by removal of mitogens and/or the addition of specific
of a single cell seeded in a sterile microtiter plate by lim- trophic factors (Rolletschek et al., 2004). Embryonic stem
iting dilution as used in the cloning of hybridoma cell cells (ESCs) have the potential to differentiate into any
lines. Thus, cell lines can be cloned to exhibit a specific cell type whereas progenitor cells are already committed
trait (e.g., high levels of specific receptors, drug resis- to follow a specific developmental pattern. Alternatively,
tance, etc.). While a homogeneous response to toxin treat- progenitor cell lines can be established; e.g., neural pro-
ment might then be expected from such a cell line, there genitor cells can be induced to differentiate into a cocul-
is the risk of losing other features of a more heteroge- ture of neuronal and glial cell types, simulating the early
neous population. In some cases, clones of transfected stages and cell cell interactions of neural development
cell lines provide useful tools for mechanistic studies in vivo (Breier et al., 2010). However, while stem cell
of cell differentiation and/or toxicity. For example, some cultures may represent excellent in vitro models for study-
cell cultures have been transfected with certain cyto- ing developmental toxicity, they are unlikely to exhibit
chrome P450 transgenes to make them metabolically identical cell cell interactions and growth patterns as
more competent during long-term studies (Tzanakakis those in mature adult tissue.
et al., 2002). One way to address this issue may be to develop a
Primary cultures are derived by a combination of postmitotic system containing predifferentiated cells prior
mechanical and enzymic disruption of the tissue of inter- to the addition of toxin. However, this issue may be better
est, which releases a collection of cells that most closely addressed by using organotypic cultures. In this case, tis-
resembles the tissue of origin both genetically and in sue slices (typically 200 μm thickness) are cut from fresh
terms of cell heterogeneity. It is then possible to enrich tissue on a microtome, then subsequently rinsed and cul-
specific cell types by using either selective culture media tured in growth medium with agitation, as discussed
and/or cell growth inhibitors, immunomagnetic beads, or above. Such slices maintain the complexity of cell cell
fluorescence-activated cell sorting (FACS). If a monocul- interactions and extracellular matrix composition of the
ture is desirable (i.e., predominantly one specific cell original tissue and in some cases can survive up to several
type), the resultant culture would then need to be screened weeks (Sundstrom et al., 2005). Nevertheless, even this
for cell-specific markers to determine purity/enrichment kind of cellular system lacks the systemic interaction with
of the preparation, as a pure culture is rarely achieved. the immune and circulatory systems that would occur
Typically, this would take the form of monitoring cultures in vivo and is not, therefore, a complete substitute for
for the expression of a unique morphological or molecular in vivo testing. However, using cell cultures as part of
trait of the desired cell type to determine the level of a tiered system of increasing complexity from in vitro to
enrichment. For example, expression of glial fibrillary in vivo measurements would improve throughput, decrease
protein and aster-like morphology would be good markers costs and allow drastic reduction in the use of live animals
for astrocytic glial cells. Cells may be cultured as mono- in screening compounds for potential toxic effects. Some
layers or, if a system simulating cell cell interactions of examples of in vitro toxicity testing systems currently in
the tissue of origin is required, cells may be cultured in use are indicated in Table 9.5.
suspension with mild agitation or by using a rolling cell
culture. Under these circumstances, cells form clusters,
spheroids, or reaggregates that may continue to grow/ Endpoint Determination for In Vitro
proliferate over a period of several days or weeks, making Testing Systems
them amenable to studies of long-term effects. For exam-
ple, brain reaggregates prepared in this way have been A good in vitro testing system should be sensitive but at
maintained for up to several weeks and used in studies of the same time yield low levels of false-positive and false-
pesticide toxicity (Sales et al., 2000). However, many pri- negative results. It should have endpoint measurements
mary cultures tend to be very short lived. Primary cell that: (1) show dose response relationships for a given
cultures do not necessarily exhibit identical cell cell toxin, (2) reflect and are predictive of the in vivo pattern
interaction patterns as those in the tissue of origin, and of toxicity for a given group of agents, (3) are objective
cell types that divide more rapidly could become more and reproducible, and (4) have internal controls. It is also
predominant than they would be in vivo. useful if the testing system involves rapid assays of toxic-
A more recent development has been the use of stem ity, allowing medium to high throughput analysis and
cell lines as models for developmental toxicity testing simultaneous testing of multiple compounds and/or doses.