Castilho Leda R., Moraes Angela Maria (Ed.) Animal Cell Technology: From Biopharmaceuticals to Gene Therapy
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process, such as nutrient deprivation, serum withdrawal, oxygen limita-
tion, hydrodynamic stress, accumulation of toxic metabolites, and viral
infection. In almost all cases, this overexpression prolonged cell viability.
However, eventually, the cultures displayed different phenotypes. In some
cases, the cell density increased, but in others a growth arrest was observed
(Vives et al., 2003a). Moreover, the effect of this overexpression in the
productivity of recombinant proteins is variable. In some cases, an increase
in productivity was observed, while in other cases there was no significant
effect or even a decrease in productivity (Vives et al., 2003a; Tey and
Al-Rubeai, 2004). In most cases, the level of Bcl-2 expression correlates
with the level of protection. Nevertheless, the expression of Bcl-2 has
shown little to no apoptosis protection in some cell types, as in the case of
HeLa, NS0, and insect (Sf9) cells (Vives et al., 2003a). Overexpression of
Bcl-X
L
has also been efficient in the control of apoptosis (Charbonneau
and Gauthier, 2000; Mastrangelo et al., 2000a, 2000b).
The cells probably have an intrinsic regulation mechanism for the level
of antiapoptotic proteins. In the Bcl-2 case, the regulation occurs through
the action of caspase 3, resulting in a 23 kDa fragment with proapoptotic
activity. Therefore, the overexpression of Bcl-2, and probably Bcl-X
L
,
may not be completely efficacious, because at some point these proteins
will be degraded or even modified into proapoptotic proteins. To bypass
this situation, a Bcl-2 mutant, resistant to the intrinsic cell regulation, was
expressed in CHO and BHK cells and showed improved efficacy over the
wild-type protein (Figueroa et al., 2001). Also, some viral Bcl-2 homolog
genes, such as E1B-19kDa (from adenovirus), BHRF1 (from Epstein–Barr
virus), and KSBcl-2 (from Kaposi’s sarcoma-associated herpesvirus) have
been studied, since they are resistant to the intrinsic cell regulation (Vives
et al., 2003b). Although the overexpression of Bcl-2 gene could not protect
NS0 cells from apoptosis, the expression of the E1B-19kDa analog could.
This suggests a different mechanism of action between E1B-19kDa and
Bcl-2 in inhibiting apoptosis (Mercille et al., 1999).
There are just a few studies of the use of caspase inhibitors to prevent
apoptosis. Most studies concentrate on the expression of proteins of the
IAP family (XIAP being the most noticeable) and the viral components
p35 and CrmA (Vives et al., 2003a). CrmA, encoded by the smallpox
virus, is a pseudo-substrate for serine and cysteine proteases. It inhibits
caspases 1, 8, and 10 in several cell types (Sauerwald et al., 2003). p35 is a
wide-spectrum caspase inhibitor encoded by baculoviruses, and it also
behaves as a pseudo-substrate, inhibiting caspases 1, 3, 6, 7, 8, and 10
(Zhou et al., 1998). XIAP is the most potent member of the IAP family. It
is found in the mammalian genome and is responsible for the inhibition of
caspases 3, 7, and 9 (Sauerwald et al., 2002). An increased protective effect
is found in CHO and HEK-293 cells expressing a XIAP mutant resistant
to degradation (XIAP-BIR123) when compared with the wild-type pro-
tein (Sauerwald et al., 2002).
Numerous pathways are involved in the activation of apoptosis,
although at some point, all these pathways seem to converge to a few
related ones. In order to be more efficient, molecular strategies to prevent
apoptosis should concentrate on these few steps of the apoptotic cascade
that are common to all cell types and to different apoptotic stimuli. For
172 Animal Cell Technology
this reason, research on genetic manipulation to prevent apoptosis is
focusing on the last steps of the cascade. While many strategies have
enabled a delay of the apoptotic process, so far none is capable of
completely circumventing it. This suggests that the cells may have other
mechanisms available to overcome the antiapoptotic activity of the ex-
pressed proteins. It is also possible that the heterologous gene is lost
during cellular division or that death by other mechanisms, such as
necrosis, is responsible for the inability to completely circumvent cell
death.
7.7 Conclusions and perspectives
Complex phenomena regulate cell proliferation and cell death both in vivo
and in vitro. The increasing understanding of these phenomena is serving
as a basis for studies aimed at improving the performance of biotechnolo-
gical processes based on in vitro animal cell culture. Multidisciplinary
research projects, involving groups from different disciplines, such as
biology and engineering, are advancing towards the development of
techniques to control cell proliferation and cell death, with the aim of
increasing the longevity and robustness of cells in culture. The outcome
of these studies will constitute, in the future, important tools to increase
productivity, predictability, and reliability of animal cell culture processes.
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Mechanisms of cell proliferation and cell death in animal cell culture
in vitro
17 9
8
Mathematical models for
gr owth and pr oduct
synthesis in animal cell
cultur e
Elisabeth F.P. Augusto, Manuel F. Barral, and
Rosane A.M. Piccoli
8.1 Introduction
It is extremely important from a scientific and technological point of view
to understand cell behavior under different culture conditions. This know-
ledge allows control of the processes that generate important commercial
products and establishes a rational basis to increase cell productivity and
yield.
By understanding cell behavior, it is possible to identify factors that
limit and inhibit cell growth and product synthesis. This allows improve-
ments in culture media formulation, defining adequate culture conditions
and keeping them at optimum values through process control. The
optimization of these conditions allows an increase in reaction rates and
their control, resulting in enhanced process productivity, higher final
product concentration, and increased substrate-to-product conversion
factor. This process optimization, in general, may also allow improve-
ments in product quality, insuring technical and economic viability.
The systematization and quantification of this knowledge through
mathematical equations, called mathematical modeling, relate culture con-
ditions to growth, death, nutrient consumption, and product synthesis
rates. Solving these equations allows a quantitative prediction of cell
behavior, meaning that it is possible to simulate the changes in the major
variables (such as component concentrations) with time. Even at the initial
stages of model development, before it is capable of representing the
totality of the observed phenomena, it is possible to use a model for the
simulation of different culture conditions. The capacity to predict
the dynamics of the population helps to understand the system under
investigation, because it may guide experimental planning, reduce the
number of experiments, and allow estimates of un-measured variables, as
well as providing rational criteria for process design, optimization, and
control of bioreactors.
Although animal cells have the ability to produce high value proteins
with post-translational modifications appropriate for clinical application,