Castilho Leda R., Moraes Angela Maria (Ed.) Animal Cell Technology: From Biopharmaceuticals to Gene Therapy
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4.4.1 Oxygen requirements
The effects of dissolved oxygen on mammalian cell growth and metabo-
lism have been studied extensively. It has been established that anaerobic
conditions can severely depress cell growth. At high oxygen concentra-
tions, on the other hand, oxidative damage is observed. Between these
limits, there exists an optimal dissolved oxygen range suitable for prolif-
eration of mammalian cells. Generally, a dissolved oxygen range between
30% and 60% air saturation is accepted to be optimal for cell growth,
although the sensitivity of cells to extreme dissolved oxygen levels is
known to vary. Each cell line seems to have a different optimal dissolved
oxygen range. It has been shown that established cell lines are more
tolerant to high dissolved oxygen levels. Lymphoblastoid cells, for in-
stance, achieved their highest cell yield and growth rates at high dissolved
oxygen concentrations (100%).
WI-38 cells grown at dissolved oxygen values between 16% and 85%
air saturation had similar growth parameters, such as population density,
glucose utilization, and lactate production. The growth rate decreased
greatly and metabolic rates of glucose and lactate increased four- to six-
fold when the dissolved oxygen was lowered to 5%. Similar increases in
glucose utilization and lactate production at low dissolved oxygen have
been reported for other cell lines. Less is known about the effects of
dissolved oxygen on glutamine metabolism. An increase in glutamine
consumption rate at very low and very high oxygen concentrations has
been reported. Effects of oxygen were also reported on ammonia produc-
tion from glutamine (Ozturk and Palssom, 1990).
The murine hybridoma CC9C10 was grown at steady state, under
serum-free conditions in continuous culture, at dissolved oxygen concen-
trations in the range of 10–150% of air saturation. There was a require-
ment for cellular adaptation at each stepwise increase in dissolved oxygen.
Adaptation to a dissolved oxygen concentration of 100% was associated
with an increase in the specific activities of glutathione peroxidase
(18-fold), glutathione S-transferase (11-fold), and superoxide dismutase
(6-fold), which are all known antioxidant enzymes. At dissolved oxygen
concentrations above 100%, the activities of glutathione peroxidase and
glutathione S-transferase decreased, possibly as a result of inactivation by
reactive oxygen radicals. The increase in dissolved oxygen concentration
caused changes in energy metabolism. The specific rate of glucose uptake
increased at higher dissolved oxygen concentrations, with a higher propor-
tion of glucose metabolized anaerobically. It was shown that the flux of
glucose through glycolysis and the pentose phosphate pathway increased,
whereas the flux through the tricarboxylic acid cycle decreased at high
dissolved oxygen concentrations (Jan et al., 2000).
Acute exposure of Chinese hamster ovary (CHO) cells or human
bladder cancer MGH-U1 cells to hypoxia plus low pH (6.5–6.0), was
cytotoxic in a time- and pH-dependent manner. Inhibition of glycolysis
by incubation of CHO cells under hypoxic conditions in the absence of
glucose at pH 7.0 led to a larger fall in cellular ATP and energy charge. A
decrease in energy charge of the cells may contribute to loss of viability,
but additional mechanisms appear to be involved (Rotin et al., 1986).
102 Animal Cell Technology
Activities of enzymes involved in glycolysis, the pentose phosphate
pathway, the TCA cycle, and glutaminolysis have been determined in the
mouse myeloma SP2/0.Ag14, when the cells were grown on IMDM
medium with 5% serum in steady-state chemostat culture under different
levels of oxygenation. The specific rates of glucose consumption and
lactate production were highest at low oxygen supply. Under low oxygen
conditions, the specific production of ammonia increased and the con-
sumption pattern of amino acids showed large changes compared with the
control culture. The levels of glycolytic enzymes were elevated under
conditions of low oxygen supply (Vriezen et al., 1997).
Not only does the oxygen level affect metabolism, but also metabolism
affects oxygen uptake rates, making a tight control of oxygen concentra-
tion necessary when the composition of the culture medium is changed.
4.4.2 Carbon dioxide
Carbon dioxide is one of the end products of mammalian cell metabolism,
produced in different metabolic steps. In some reactions, carbon dioxide
can be used as a substrate, such as the carboxylation of pyruvate to
oxaloacetic acid. The importance of carbon dioxide is also related to its
generalized use in cell culture technology for pH regulation, in equili-
brium with medium bicarbonate. The influence of carbon dioxide genera-
tion by cell metabolism can be especially relevant in industrial
applications, operating with systems optimized to provide an efficient
oxygen supply and to reach high cell densities. In such systems, the
metabolically produced carbon dioxide may increase, with an elevated
partial pressure, which can lead to reduced cell viability, decreased specific
production rates, and polysialylation rates (Zanghi et al., 1999).
4.4.3 Temperature
Standard temperatures are usually defined for every known cell line, for
example, mammalian cell lines are usually cultured at 378C and insect cell
lines at 288C. However, studies have demonstrated that temperature
modifications can lead to changes in certain parameters, such as increased
productivity of recombinant proteins or decreased byproduct generation.
Several studies have shown environmental control strategies to affect
protein glycosylation and carbon metabolism, as well as cell growth and
cell death. Experiments suggest that reduced culture temperature results in
higher viability and shear resistance, decreased specific growth rate,
limited release of waste products, and reduced glucose/lactate metabolism.
There are several reports on the effects of the culture temperature on cells
producing recombinant proteins (Bollati-Fogolin et al., 2005). In some
cases reduced temperatures were associated with improved cellular pro-
ductivity, and prolonged cellular viability in culture (Rodriguez et al.,
2005). However, Weidemann et al. (1994) described that a temperature
below 378C reduced the cell growth and glucose consumption of a
recombinant BHK cell line but did not affect the cellular productivity of
recombinant antithrombin III.
Cell metabolism and its control in culture 103
4.4.4 pH
Ideally, the pH should be near 7.4 at the initiation of a culture and remain
above a value of 7.0 during the culture, although some hybridomas appear
to prefer lower pH. A pH below 6.8 is usually inhibitory to cell growth.
Factors affecting the pH stability of the medium are buffer capacity,
volume of headspace, and glucose concentration.
As described in Chapter 2, the usual normal buffer system in culture
media is the carbon dioxide-bicarbonate system, analogous to that in the
blood. Modifications in the medium pH produce changes to the intracellu-
lar pH value, with modifications to enzyme activities.
4.5 Conclusions
Mammalian cell culture is a technology used for the production of
recombinant proteins of therapeutic use, as they can secrete proteins with
post-translational modifications similar to those present in human pro-
teins. The most important advantages of this capacity of the mammalian
cell lines are that they secrete a protein with the similar characteristics to
the original protein, so that the protein can be used for human treatment
without generating immunological responses.
As the productivity of mammalian cell lines is lower compared with that
of other production systems (bacteria, insect cells, etc.), it is desirable to
improve the productivity per cell or the total cell yield per unit volume of
culture. An increase in the specific productivity could involve enhance-
ment at the genetic level by gene amplification or by the addition of an
inducer to enhance the transcription of a gene. However, knowledge of
mammalian cell metabolism helps in the development of strategies for
productivity enhancement, as changes in the culture conditions can affect
both metabolism and productivity.
The cell yield can be improved by a full understanding of the physico-
chemical environment of the cells during growth and maintenance. It is
now becoming clear that cells in culture are highly adaptable and may be
maintained under conditions that are substantially different from their
normal environment in vivo. Intracellular enzyme activity levels can
change considerably and such changes can alter fluxes through metabolic
pathways, which may reflect the optimal utilization of the nutrients made
available in the culture medium (Crabtree and Newsholme, 1987; Glacken,
1988; Butler et al., 1989).
Thus, knowledge of cell physiology is an important prerequisite for
further directed metabolic optimization and the investigation of associated
cellular phenomena.
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110 Animal Cell Technology
5
Cultur e media for animal
cells
A
ˆ
ngela Maria Moraes, Ronaldo Zucatelli Mendonc¸a, and
Claudio Alberto Torres Suazo
5.1 Introduction
Effective in vitro maintenance and growth of animal cells requires culture
conditions similar to those found in vivo with respect to temperature,
oxygen and carbon dioxide concentrations, pH, osmolality, and nutrients.
Within normal tissue in vivo, animal cells receive nutrients through blood
circulation. For growth in vitro, animal cells require an equivalent supply
of a complex combination of nutrients. For this reason, the first attempts
in animal cell culture were based on the use of biological fluids such as
plasma, lymph and serum, as well as on extracts from embryonic-derived
tissue.
Medium composition is one of the most important factors in the culture
of animal cells. Its function is to provide appropriate pH and osmolality
for cell survival and multiplication, as well as to supply all chemical
substances required by the cells that they are unable to synthesize
themselves. Some of these substances can be provided by a culture medium
consisting of low molecular weight compounds, known as basal media.
However, most basal media fail to promote successful cell growth by
themselves and require supplementation with more complex and chemi-
cally undefined additives such as blood serum.
Many attempts have been made to develop culture media that do not
need this type of supplementation, that is, serum-free media formulations.
These serum-free media present several practical advantages. However, the
formulations that have been developed are specific for certain cell types
and a universal culture medium suitable for the culture of all animal cell
types seems, so far, unreachable.
While some culture media are formulated to promote cell multiplication
(growth media), others only maintain cell structural and metabolic integ-
rity (maintenance media), but do not stimulate cell division. Media
prepared with highly purified compounds and with known composition
are designated chemically defined media. These are particularly attractive
for biopharmaceutical production, because they are less vulnerable to
contamination and quality control is easier. Nevertheless, these media can
be expensive.
A complete medium for the culture of animal cells can be considered to
have two distinct parts. The first supplies basic cell needs of nutrients,