Castilho Leda R., Moraes Angela Maria (Ed.) Animal Cell Technology: From Biopharmaceuticals to Gene Therapy

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formation. The combination of a low incorporation of acetyl-CoA into

the cycle and the outﬂow of citrate depletes the metabolites of the TCA

cycle, which need to be replenished to satisfy requirements for energy

generation, since the ATP production by glycolysis is low. Replenishment

may occur at the level of Æ-ketoglutarate. The primary role of glutamine is

to supply this intermediate to keep the operation of the cycle. In this way,

glutamine is a major source of carbon and energy. Other amino acids and

lipids also participate in the supply of intermediates to the TCA cycle

through anaplerotic reactions (Figure 4.2).

In most media formulations for mammalian cell lines, glucose is in-

cluded at an initial concentration of 10–25 mM, which decreases to around

half the concentration level within the period of a batch culture. The

production of lactic acid may lower the pH of the culture and this may

decrease the cellular growth rate. However, if the culture is appropriately

buffered then the accumulated lactate levels typically found in a batch

culture should not cause any growth inhibition (Hassell et al., 1991).

As glucose is metabolized at a far greater rate than it is needed to

maintain viability, it is better to supplement it during the culture than to

Phosphoenolpyruvate

PYRUVATE

KINASE

PyruvateLactate

LACTATE DEHYDROGENASE

ALANINE AMINO

TRANSFERASE

Alanine

Oxalacetate

Malate

Citrate

PYRUVATE

CARBOXYLASE

MALIC

ENZYME II

PYRUVATE

DEHYDROGENASE

PHOSPHOENOL-

PYRUVATE

CARBOXYKINASE

GLUTAMATE

HYDROGENASE

GLUTAMINASE

TCA

CYTOSOL

MITOCHONDRIA

Acetyl CoA

α-ketoglutarate

Glutamate

Glutamane

Figure 4.3

Enzymes that link glycolysis and the tricarboxylic acid (TCA) cycle. Dashed arrows indicate reduced

or suppressed enzymatic activity.

82 Animal Cell Technology

increase the initial concentration. This will limit the accumulation of

lactate in the culture. Nevertheless, the cell concentration and the protein

productivity may be affected by glucose concentration. Meijer and van

Dijken (1995) have reported the effects of the glucose supply on growth

and metabolism of an SP2/0 derived recombinant myeloma cell line in a

chemostat culture with Iscove’s Modiﬁed Dulbecco’s Medium (IMDM)

medium. Lowering glucose concentration of the feed medium from 25.0 to

1.4 mM resulted in a decrease of steady-state viable cell concentration,

whereas viability remained above 90%. Sun and Zhang (2001a) have

reported the effects of the glucose concentration on batch cultures of

recombinant CHO (Chinese hamster ovary) cells. They demonstrated that

the accumulated concentration of erythropoietin (EPO) increased with the

increase of glucose concentration from 8.9 to 17.9 mM, and decreased with

its further increase from 17.9 to 49.6 mM. They concluded that there is an

optimal glucose concentration for the enhancement of EPO expression by

these recombinant CHO cells. They also demonstrated that the yield

coefﬁcient of lactate from glucose increased with the initial glucose

concentrations from 8.9 up to 17.9 mM, and then kept constant above

17.9 mM.

An alternative is to substitute glucose by other hexoses, like galactose or

fructose, or by disaccharides, like maltose. Galactose is converted into

galactose-6-phosphate, a reaction catalyzed by hexokinase, and then it is

transformed into glucose-6-phosphate, an intermediate of glycolysis.

Fructose can also be phosphorylated to fructose-6-phosphate by hexoki-

nase. Fructose can also enter the glycolysis pathway at the level of

glyceraldehyde-3-phosphate, via the fructose-1-phosphate pathway. As

maltose is a disaccharide of glucose, it is slowly hydrolyzed into glucose

molecules and then transported into the cell and metabolized. The meta-

bolism of alternative carbohydrate sources provides different amounts of

energy. The ﬂux through glycolysis may be slower due to a low afﬁnity of

the hexokinase enzyme for galactose and fructose or due to the slow

hydrolysis of maltose. Thus, substitution of glucose with fructose, galac-

tose, or maltose may decrease the rate of production of lactic acid (Butler

and Christie, 2004).

The reduced glycolytic rate and formation of lactic acid may result in a

slower growth rate (Grifﬁths, 2000). Duval et al. (1992) cultured a mouse

hybridoma cell line (VO 208) in batch/fed-batch cultures in a medium

supplemented with fructose instead of glucose and demonstrated an in-

crease of the lifespan of the culture and an enhancement in the antibody

secretion. However, it has been reported that complete glucose substitution

by other hexoses can alter product glycosylation (Paredes et al., 1999).

Another strategy to improve the efﬁciency of central carbon metabolism

and to reduce lactate accumulation is metabolic engineering. Increasing

the ﬂux of glucose into the TCA cycle can improve the efﬁciency of

glucose for energy metabolism by enhanced formation of ATP. Weide-

mann et al. (1994) have reported that the introduction of a cytosolic

pyruvate carboxylase derived from Saccharomyces cerevisiae into BHK-21

cells enabled cells to transfer glycolysis-derived pyruvate into malate,

which then entered the TCA cycle for complete oxidation. As a result,

higher yields of recombinant EPO were achieved by the BHK cells.

Cell metabolism and its control in culture 83

4.2.2 Glutamine

Glutamine is the major source of energy, carbon, and nitrogen for

mammalian cells. It plays two important functions: it acts as an energy

donor and plays a critical role in nitrogen metabolism, acting as a collec-

tion point for amino groups. For example, in the cytosol of hepatocytes,

amino groups from most amino acids are transferred to Æ-ketoglutarate to

form glutamate, which enters the mitochondria and gives up its amino

group to form ammonium. Excess ammonium generated in most tissues

other than the liver is converted to the amide nitrogen of glutamine.

Glutamine and glutamate, or both, are present in higher concentrations

than other amino acids in most tissues or biological ﬂuids.

Glutamine is transported into the cytosol of the cell by means of two

kinds of transporters: Na

-dependent and Na

-independent. Once in the

cytosol, glutamine must be transported into the mitochondria, where it is

metabolized.

The metabolic pathway of glutamine, called glutaminolysis, begins with

an enzymatic reaction catalyzed by glutaminase, which converts glutamine

into glutamate with ammonium removal. Then, glutamate is converted

into Æ-ketoglutarate by the action of the glutamate dehydrogenase, liberat-

ing another ammonium ion (Figure 4.4). This Æ-ketoglutarate enters

directly to the TCA cycle (Figure 4.2).

In culture media for mammalian cells, together with the carbohydrate

source, glutamine is the most abundant source of reduced carbon. Similar

to glucose, glutamine is also consumed by mammalian cells at a high rate,

particularly if the glutamine concentration is higher than the minimal

needs for maintaining cell viability. This leads to the unwanted accumula-

tion of ammonium ions, a toxic byproduct, in addition to an inefﬁcient

use of glutamine.

Glutamine is normally included at a concentration of 1–5 mM, which is

a signiﬁcantly higher concentration than that of any other amino acids.

Glutamine is an important precursor for the synthesis of purines, pyrimi-

dines, amino sugars, and asparagine. However, glutamine also has an

important role as substrate for the TCA cycle (Butler, 2004).

For some mammalian cells, glutamine is the main source of energy. It

was observed in an antibody-secreting murine hybridoma (CC9C10)

culture that, after 2 days of exponential growth in batch culture in a

medium containing 20 mM glucose and 2 mM glutamine, the glutamine

content of the medium was completely depleted, whereas the glucose

content was reduced to only 60% of the original concentration. Petch and

Butler (1994) demonstrated that glucose is normally metabolized via

GlutamateGlutamine

GLUTAMINASE

NADP

⫹

GLUTAMATE

DEHYDROGENASE

NADPH

H⫹

⫹

α-ketoglutarate

Figure 4.4

Initiation of the glutamine metabolism pathway.

84 Animal Cell Technology

glycolysis (. 96%), the PPC (3.6%), and the TCA cycle (0.6%). Gluta-

mine, on the other hand, is partially oxidized via glutaminolysis to alanine

(55%), aspartate (3%), glutamate (4%), lactate (9%), and CO

(22%)

(Petch and Butler, 1994). Fitzpatrick et al. (1993) demonstrated that, in a

murine B-lymphocyte hybridoma (PQXB1/2) batch culture, glutamine

was completely depleted, but glucose was partially depleted to only 50%

of its original concentration when the cells reached a stationary phase

following exponential growth. This suggests that glutamine is the major

contributor of cellular energy. Vriezen et al. (1997) cultured two different

cell lines (MN12, a mouse–mouse hybridoma, and SP2/0-Ag14, a mouse

myeloma) in steady-state chemostat cultures fed with IMDM medium

with 5% serum and glutamine concentrations ranging from 0.5 to 4 mM.

The culture proﬁles showed that glutamine was the growth-limiting

substrate in this concentration range. Furthermore, they showed that

excess glutamine gives rise to high consumption rates: in glutamine-limited

cultures, the speciﬁc rates of ammonia and alanine production were low

compared with glutamine-rich cultures containing 4 mM glutamine in the

feed medium.

The preferential utilization of glutamine as an energy source is cell-

dependent. Maranga and Goochee (2006) have demonstrated that

PER.C6

cells fall into a minor category of mammalian cell lines for

which glutamine plays a minor role in energy metabolism.

Although glutamine is used as a major substrate for the growth of

mammalian cells in culture, it suffers from some disadvantages. The meta-

bolic deamination of glutamine leads to ammonia, which accumulates in

the culture up to 2–4 mM and can be inhibitory to cell growth. The

problem of ammonia accumulation is made worse by the fact that

glutamine may decompose spontaneously to produce ammonia in the

culture medium at a rate of 0.1 mM per day at 378C. The extent of growth

inhibition by ammonia is greater at higher pH levels and is cell line-

dependent.

The accumulation of ammonia from glutamine can be decreased by a

continuous feed of a low concentration of glutamine into the culture.

Ljunggren and Ha

ggstro

m (1992) cultured the murine myeloma cell line

Sp2/0-Ag 14 in an ordinary batch culture and in a glutamine-limited fed-

batch culture. In batch culture, the overﬂow metabolism of glutamine led

to excess production of ammonium and the amino acids alanine, proline,

ornithine, asparagine, glutamate, serine, and glycine. In the fed-batch

culture, glutamine limitation halved the cellular ammonium production

and reduced the ratio of ammonium/glutamine. The excess production of

alanine, proline, and ornithine was reduced by a factor of 2–6, while

asparagine was not produced at all. They demonstrated that essential

amino acids were used more efﬁciently in the fed-batch culture as judged

by the increase in the cellular yield coefﬁcients in the range of 1.3–2.6

times for 7 of the 11 amino acids consumed. Altogether, this leads to a

more efﬁcient use of the energy sources glucose and glutamine, as revealed

by an increase in the cellular yield coefﬁcient for glucose by 70% and for

glutamine by 61%, as well as a reduction in the generation of ammonium.

Maranga and Goochee (2006) have reported that, when PER.C6

cells

were cultured in suspension in a 2-liter serum-free bioreactor at a con-

Cell metabolism and its control in culture 85

trolled glutamine concentration of 0.25 mM, the consumption rate of

glutamine and the production rate of ammonium were reduced by

approximately 30%, with respect to the same culture with a higher

glutamine concentration. Furthermore, the consumption rate of alanine

and the consumption rate of non-essential amino acids were reduced by

85% and 50%, respectively. The fed-batch control of glutamine also

reduced the overall accumulation of ammonium ion by approximately

50% by minimizing the spontaneous thermal degradation of glutamine.

In order to reduce the ammonium production, substitution of glutamine

by an alternative substrate such as glutamate may be possible. However, if

glutamate is used, a period of adaptation is required during which the

activity of glutamine synthetase and the rate of transport of glutamate

both increase, since glutamate has a low efﬁciency transport system. The

cell yield increases when ammonia accumulation is decreased following

culture supplementation with glutamate rather than glutamine. However,

some cell lines fail to adapt to growth in glutamate (McDermott and

Butler, 1993). The cell line HL-60 was adapted to growth in glutamine-

deﬁcient medium by stepwise glutamine deprivation. The successful

adaptation of different cell lines to non-ammoniagenic medium has been

described by Hassell and Butler (1990). They replaced glutamine by either

glutamate or Æ-ketoglutarate. A mole to mole substitution of glutamine by

glutamate was successful for a McCoy cell line and led to normal growth

rates after approximately 10 days. Cell yield was increased by 17%,

ammonia accumulation was reduced by 70%, and glucose consumption

and lactate production both decreased by more than 70%. A BHK and a

Vero cell line had to be slowly adapted from an initially high glutamate

concentration. The MDCK cell line could not be adapted to growth on

glutamate. The authors proposed that the glutamate uptake, and not the

glutamine synthetase activity, is responsible for the ability of a given cell

line to grow in a glutamine-free medium (McDermott and Butler, 1993).

An alternative to cell adaptation is metabolic engineering. Cells trans-

formed with the glutamine synthetase gene can grow in media supplemen-

ted with glutamate instead of glutamine, with a direct elimination/

reduction of ammonia generation, either by glutamine decomposition or

metabolism (Paredes et al., 1999). Bell et al. (1992, 1995) successfully

obtained a murine hybridoma cell line that could grow in the complete

absence of glutamine by transformation with the glutamine synthetase

gene. Ammonia concentrations in the medium of batch cultures of these

cells were below detection levels. This cell line could not be adapted to

glutamine-free growth even in the presence of elevated levels of glutamate,

so metabolic engineering was the unique alternative.

Another strategy for the reduction of ammonia accumulation can be the

use of glutamine-containing dipeptides, which hydrolyze slowly in the

culture (Butler and Christie, 2004). The supplementation of a glutamine-

free medium with dipeptides containing glutamine allows a reduction in

the rate of ammonium generation, but this requires the presence of

dipeptidases, which may be produced by the cells and may be released into

the medium. Christie and Butler (1994) grew a murine hybridoma

(CC9C10) in media containing the dipeptides alanyl-glutamine (Ala-Gln)

or glycyl-glutamine (Gly-Gln) as a substitute for glutamine. They

86 Animal Cell Technology

obtained high cell yields in the presence of 6 mM Ala-Gln or 20 mM

Gly-Gln, with the ﬁnal cell yield in Gly-Gln 14% higher than in Gln. The

higher concentration of Gly-Gln was necessary for cell growth because of

the presence of a peptidase (in the cytosolic fraction of the cells) with a

lower afﬁnity for Gly-Gln. Monoclonal antibody productivity was com-

parable in Gln, Ala-Gln, or Gly-Gln. Substrate utilization and metabolism

was affected by the presence of the dipeptides, particularly with Gly-Gln.

The speciﬁc consumption rates of glucose and six amino acids were

reduced and the accumulation of ammonia and lactate was signiﬁcantly

lower.

The replacement of glutamine (2 mM) by pyruvate (10 mM) supported

cell growth of several adherent cell lines (MDCK, BHK-21, CHO-K1) in

serum-containing and serum-free media, without adaptation for at least 19

passages and with no reduction in growth rate. Even at very low levels of

pyruvate (1 mM), MDCK cells grew to conﬂuence without glutamine or

accumulation of ammonia. Also glucose uptake was reduced, which

resulted in lower lactate production. Amino acid proﬁles from the cell

growth phase for pyruvate medium showed a reduced uptake of serine,

cystine, and methionine, an increased uptake of leucine and isoleucine, and

a higher release of glycine compared with glutamine medium (Genzel

et al., 2005).

4.2.3 Amino acids

Amino acids are a class of biomolecules that make a signiﬁcant contribu-

tion to the generation of metabolic energy. The fraction of metabolic

energy obtained from amino acids varies greatly with the type of cell and

with metabolic conditions.

The transport of amino acids into mammalian cells can be regulated by

nutritional, hormonal, or other environmental factors or by changes with-

in cells like transformation. The intracellular or extracellular concentration

of amino acids probably has the most profound inﬂuence on the efﬁciency

or capacity of transport into animal cells.

The TCA cycle functions as a major route for the synthesis and

oxidation of most amino acids and is the primary route through which

amino acid carbon ﬂows in the synthesis of other small molecules (Figure

4.5).

Amino acids are a vital constituent of all cell culture media. They are

normally added as deﬁned components to cell culture medium. The

importance of amino acids in synthetic media for in vitro growth of

mammalian cells has long been recognized, as both a nitrogen donor and a

carbon source. Studies on the rates of amino acid uptake have shown

generally that glutamine is the most rapidly consumed, followed by lysine,

leucine, and isoleucine (Roberts et al., 1976). The nutritional requirement

for a certain metabolite, however, may also be inﬂuenced by the cell

population density. For example, serine, cystine, glutamine, and asparagine

have been shown to be required at low, but not at high, cell densities

(Eagle and Piez, 1962). This occurs in situations when the metabolite is

utilized in amounts that exceed the biosynthetic capacity of the cell. The

critical population density occurs at the minimum effective intracellular

Cell metabolism and its control in culture 87

level, before the cells die of the speciﬁc deﬁciency. At high cell densities,

however, the cell culture medium may require supplementation with extra

amino acids, to prevent their depletion (Doyle and Grifﬁths, 1998). The

uptake of leucine, isoleucine, and methionine is closely related to the cell’s

position in the cell cycle and is regulated by growth factors (such as

insulin) in mammalian cells (Doverskog et al., 1997).

Higher eukaryotic cells have lost the ability to synthesize a number of

amino acids. These amino acids are generally called essential amino acids,

while those that can be synthesized are called non-essential. However, this

nomenclature is very misleading for two reasons. First, some of the non-

essential amino acids are in fact very essential in that they are required for

synthesis of nucleotides (glycine, aspartate, and glutamine). The reason

why the ability to synthesize these amino acids has been retained may well

be that they are indispensable. Secondly, the capability to synthesize them

Pyruvate

Glycine

Serine

Alanine

Cystine

Threonine

Lysine

Acetyl CoA Acetoacetate

3-hydroxy 3-methylglutaryl-CoA-

Oxalocetate

Fumarate

Succinyl CoA

TCA

Leucine

Phenylalanine

Tyrosine

Tryptophan

Asparagine

Aspartate

Tyrosine

Phenylalanine

Valine

Methionine Isoleucine

Threonine

α-ketoglutarate

Proline

Histidine

Glutamate

Glutamine

Ornithine

Arginine

Figure 4.5

Points at which amino acids enter or exit the tricarboxylic acid (TCA) cycle in mammalian cells.

88 Animal Cell Technology

may be conditional depending on the nutritional situation, the prolifera-

tive status, and cell line-speciﬁc properties (Doverskog et al., 1997). The

early work of Eagle (Eagle, 1955) demonstrated a need for 12 amino acids

to support the proliferation of strain L mouse ﬁbroblasts in medium

containing 0.25–2% dialyzed horse serum. Cells would die within 1–

3 days in the absence of any one of the 12 amino acids. Glutamine was

later added to this list (Eagle, 1959), which also includes arginine, cystine,

histidine, isoleucine, leucine, lysine, phenylalanine, methionine, threonine,

tryptophan, tyrosine, and valine.

The rates at which these and other amino acids are utilized or produced

can vary dramatically between cell lines. The relative concentration of

amino acids and serum in the culture medium and other conditions of the

culture environment will also inﬂuence the rates of utilization or produc-

tion of speciﬁc amino acids.

Amino acids, whose carbon skeleton can be synthesized de novo in

mammalian cells, include serine, glycine, alanine, aspartate, asparagine,

and in principle glutamate and glutamine. The biosynthesis of amino acids

is closely linked to the central intermediary metabolism, occurring directly

from intermediates of glycolysis or TCA, in one or a few steps. The key

enzymes involved are transaminases (Meister, 1955). Although most cells

possess glutamate dehydrogenase, it is doubtful if there is any signiﬁcant

net synthesis of glutamate through this enzyme in cultured cells. The

allosteric regulation of glutamate dehydrogenase involving up-regulation

by GDP (guanosine 5’-diphosphate) and ADP, and inhibition of enzyme

activity by GTP and ATP indicates that it has a catabolic function, that is,

the enzyme is activated when cells need amino acids for energy production

(Mehler, 1982). Glutamine, proline and ornithine are all synthesized from

glutamate (Doverskog et al., 1997).

The amino acids that can be synthesized by a cell depend upon the

strain-speciﬁc proﬁle of biosynthetic enzymes. For example, BHK and

CHO cells are capable of glutamine synthesis (Street et al, 1993; Neer-

mann and Wagner, 1996), while hybridoma and myeloma cells that do not

possess glutamine synthetase are not (Bebbington et al., 1992). Another

example of a strain-speciﬁc difference is the ability to synthesize glycine.

Sf9 insect cells and certain CHO cell mutants are reported to be partial

glycine auxotrophs (Appling, 1991; Tremblay et al., 1992; Chasin et al.,

1994). The explanation involves the localization of serine hydroxymethyl-

transferase. This enzyme, which converts serine to glycine and tetrahydro-

folate-bound single-carbon units, is present both in the cytoplasm and

mitochondria. The mitochondrial isoenzyme activity may be absent in

these partial auxotrophs, which are self-supporting in single-carbon units

through the cytoplasm enzyme activity, but they need glycine from the

medium for protein synthesis. In contrast to mammalian cells, insect cells

are much more ﬂexible in their amino acid metabolism (Ferrance et al.,

1993), some cell lines being capable of synthesizing many more amino

acids (Mitsuhashi, 1982), including glutamine, glutamate, and aspartate

simultaneously (O

hman et al., 1996).

The capability of synthesis of a certain amino acid may be conditional,

and depends on the availability of carbon precursors and nitrogen donors.

For example, a glutamine-free medium for mammalian cells may have to

Cell metabolism and its control in culture 89

be supplemented with aspartate and/or asparagine (Bebbington et al.,

1992) to supply intracellular precursors for glutamine biosynthesis and as

a source of aspartate and asparagine, the synthesis of which may become

limited in a glutamine-free medium. Another example of conditional

biosynthesis is the formation of cystine (from methionine, the cystathio-

nine pathway) in Sf9 cells, which appears to be regulated in relation to the

proliferative status of the cells (Doverskog et al., 1997).

It has been recognized that certain cells have a speciﬁc requirement for

an amino acid, for example, serine for lymphoblastoid cells (Birch and

Hopkins, 1977). This may be due either to the inability of the cells to make

an amino acid, or because the amino acid is decomposed in the medium.

The concentration of amino acids usually limits the maximum cell concen-

tration attainable, inﬂuences cell survival and growth rate, and can affect

the synthesis of certain proteins. A too low concentration of an amino acid

can result in rapid depletion from the medium, and is thus ‘‘limiting,’’

whereas a too high concentration can be inhibitory.

Branched chain amino acids are consumed particularly rapidly by a

number of cell lines including MDCK cells (Butler and Thilly, 1982),

human ﬁbroblasts (Lambert and Pirt, 1975), mouse myeloma cells

(Roberts et al., 1976), and BHK cells (Arathoon and Telling, 1982). Miller

et al. (1989) found that serine and branched amino acids were more

extensively oxidized by hybridoma cells when the speciﬁc glutamine

utilization rate was low. The sulfur-containing amino acids, methionine

and cystine, are also rapidly consumed by cells in culture (Lambert and

Pirt, 1975; Butler and Thilly, 1982). It is suggested that a function of

glutamine uptake and glutamate formation is to allow cystine uptake by

glutamate exchange into the culture medium (Bannai and Ishii, 1988).

Certain amino acids often accumulate in the culture medium during

batch growth with surplus glucose and glutamine (Grifﬁths, 1971; Lanks

and Li, 1988; Duval et al., 1991; Ljunggren and Ha

ggstro

m, 1992). How-

ever, in glutamine-limited fed-batch cultures of myeloma cells the over-

ﬂow of asparagine, ornithine, proline, alanine, and glutamate is

considerably less than in batch cultures. The same situation exists in

hybridoma cells (Ljunggren and Ha

ggstro

m, 1995). These results can be

explained by the involvement of glutamine and glutamine-derived gluta-

mate, both in the nitrogen transfer reactions and in providing carbon

precursors. The results also highlight the close coupling between the

amino acid and energy metabolism (Doverskog et al., 1997).

One of the determinants of cell growth and survival in cultures of Sf9

cells is the transport of amino acids across the plasma membrane. Uptake

of cystine increases as a function of the cystine concentration in the

medium. However, the increased cystine uptake does not lead to an

increase in the ﬁnal cell concentration or in the growth rate. On the

contrary, there appears to be a negative inﬂuence on cell physiology as

more of the energy-yielding substrates, glucose, glutamine, and glutamate

are consumed at the higher cystine concentration. This is another example

of less tight regulation of metabolism in animal cells (Doverskog et al.,

1997).

Alanine, an end product of glutamine metabolism in many mammalian

cells (McKeenhan, 1986), is also formed by Sf9 cells. In a normal batch

90 Animal Cell Technology

culture of Sf9 cells with excess glucose and glutamine, alanine is the only

overﬂow metabolite that accumulates to signiﬁcant concentrations. The

use of substrate-limited fed-batch cultures revealed some interesting

features of Sf9 metabolism. During glucose limitation alanine formation

was totally depressed, while instead ammonium formation was triggered.

Glutamine limitation decreased alanine formation somewhat without

provoking ammonium formation, and during simultaneous glucose and

glutamine limitation, very little overﬂow metabolism occurred. These

results indicate that in Sf9 cells, glucose-derived pyruvate is the carbon

precursor for alanine and that glutamine provides the nitrogen. As in

hybridoma (Ljunggren and Ha

ggstro

m, 1995) and myeloma cells (Ljung-

grenn and Haggstrom 1992), the energy metabolism of Sf9 insect cells

becomes more efﬁcient during substrate limitation (O

hman et al., 1996).

Consequently, quantitative amino acid data are important to optimize

the formulation of cell culture media. The concentration of many amino

acids can be determined and the data used to calculate the rate of

utilization or assimilation of the individual amino acids (Doyle and

Grifﬁths, 1998).

4.2.4 Lipids

The oxidation of long-chain fatty acids to acetyl-CoA is a central energy-

yielding pathway in many organisms and tissues. In mammalian heart and

liver, for example, it provides as much as 80% of the energetic needs under

all physiological conditions (Lodish et al., 2003).

Lipids have several important functions in animal cells, which include

serving as structural components of membranes and as a stored source of

metabolic fuel (Griner et al., 1993). Eukaryotic cell membranes are

composed of a complex array of proteins, phospholipids, sphingolipids,

and cholesterol. The relative proportions and fatty acid composition of

these components dictate the physical properties of membranes, such as

ﬂuidity, surface potential, microdomain structure, and permeability. This

in turn regulates the localization and activity of membrane-associated

proteins. Assembly of membranes necessitates the coordinate synthesis

and catabolism of phospholipids, sterols, and sphingolipids to create the

unique properties of a given cellular membrane. This must be an extremely

complex process that requires coordination of multiple biosynthetic and

degradative enzymes and lipid transport activities.

Saturated fatty acids or unsaturated fatty acids, such as oleic acid (18:1,

n-9), can be synthesized by normal mammalian cells that posses elongation

and desaturation enzymes (Rosenthal, 1987). However, the polyunsatu-

rated fatty acids of the n-3 and n-6 group, such as linoleic acid (18:2, n-6)

or linolenic acid (18:3, n-3), are essential nutrients for animals because they

are precursors for the synthesis of eicosanoid hormones such as prosta-

glandins (Needleman et al., 1986).

The enzymes of fatty acid oxidation in mammalian cells are located in

the mitochondrial matrix. The fatty acids with chain lengths of 12 or fewer

carbons enter the mitochondria without the help of membrane transpor-

ters. Those with 14 or more carbons, which constitute the majority of fatty

acids obtained in the diet or released from adipose tissue, cannot pass

Cell metabolism and its control in culture 91