Castilho Leda R., Moraes Angela Maria (Ed.) Animal Cell Technology: From Biopharmaceuticals to Gene Therapy
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salts, and pH control, while the second comprises a set of supplements that
provide other cell needs, allowing cell growth in the basal medium. A
nutrient can be defined as a chemical substance that enters into the cell and
is used as a structural component, such as a substrate for cell biosynthesis
or energy metabolism, or as a catalyst in metabolic processes. Additional
compounds required for cell proliferation can be considered as supple-
ments, including all undefined additives such as serum and other biological
fluids.
Media frequently employed in the culture of continuous mammalian
cell lines include: Eagle’s medium, MEM (Eagle, 1959); Eagle’s medium
modified by Dulbecco, DMEM (Dulbecco and Freeman, 1959); RPMI
1640 medium (Moore et al., 1967); CMRL
TM
1066 medium (Parker et al.,
1957); and Ham’s F12 medium (Ham, 1965). For the cultivation of
adherent continuous cell lines the following basal media are suitable:
CMRL
TM
1066, MCDB 411, DMEM. F12, MCDB 301, and IMDM. For
non-transformed cells, the media DMEM, IMDM, MCDB 104, 105, 202,
401, and 501 are suitable (Freshney, 1992). Each of these basal formula-
tions may be supplemented with serum or other specific proteins.
For insect cells the following basal media can be used: Grace’s, TC 100,
TNM-FH, D22, Schneider, and M3. These media normally require supple-
mentation with fetal bovine serum. Alternatively, different serum-free
media are available for insect cells, such as Sf900II, Ex-Cell
1
400, 405, and
420, Express Five
1
SFM, Insect-XPRESS
TM
, HyQ SFX-Insect
TM
, and
IPL 41. These have the advantage of higher reproducibility and lower cost
when compared with serum-supplemented basal media (Ikonomou et al.,
2003).
For comparison purposes, Table 5.1 shows the composition of DMEM
(one of the most versatile medium formulations, typically employed in the
culture of mammalian cells) and Schneider’s medium (used for diptera
insect cells, especially Drosophila melanogaster). A total of 34 different
components are present in DMEM, although some minor variations in
composition may occur between suppliers.
Table 5.1 Composition of minimum Eagles’s medium modified by Dulbec-
co, DMEM, used for mammalian cells, and of Schneider’s medium, used in the
culture o f dipteran cells
Component Concentration in medium
(mg/ml)
DMEM Schneider
Amino acids
L-Alanine – 500,0
Glycine 30.0 250.0
L-Aspartic acid – 400.0
L-Glutamic acid – 800.0
L-Arginine – 600.0
L-Arginine hydrochloride 84.0 –
L-Cysteine – 60.0
L-Cystine 48.0 –
112 Animal Cell Technology
Table 5.1 (continued)
Component Concentration in medium
(mg/ml)
DMEM Schneider
L-Cystine dihydrochloride – 26.7
L-Glutamine 584.0 1800.0
L-Histidine – 400.0
L-Histidine hydrochloride.H
2
O 42.0 –
L-Isoleucine 105.0 150.0
L-Leucine 105.0 150.0
L-Lysine hydrochloride 146.0 2060.8
L-Methionine 30.0 150.0
L-Phenylalanine 66.0 –
L-Proline – 1700.0
L-Serine 42.0 250.0
L-Threonine 95.0 350.0
L-Tryptophan 16.0 100.0
L-Tyrosine 72.0 –
L-Tyrosine 2Na – 72.0
L-Valine 94.0 300.0
Vitamins
Choline chloride 4.0 –
D-Pantothenate Ca 4.0 –
Folic acid 4.0 –
i-Inositol 7.2 –
Nicotinamide 4.0 –
Pyridoxine hydrochloride 4.0 –
Riboflavin 0.4 –
Thiamine hydrochloride 4.0 –
Inorganic salts
Calcium chloride (CaCl
2
) 200.0 600.0
Ferric nitrate (Fe(NO
3
)
3
.9H
2
O) 0.1 –
Magnesium sulfate heptahydrate (MgSO
4
.7H
2
0) 200.0 –
Magnesium sulfate – 1810.0
Potassium chloride (KCl) 400.0 1600.0
Monobasic potassium phosphate (KH
2
PO
4
) – 450.0
Sodium bicarbonate (NaHCO
3
) 3700.0 400.0
Sodium chloride (NaCl) 6400.0 2100.0
Monobasic sodium phosphate monohydrate
(NaH
2
PO
4
.H
2
O)
125.0 –
Monobasic sodium phosphate (NaH
2
PO
4
) 700.0
Other components
D-Glucose (dextrose) 4500.0 2000.0
D-Threalose – 2000.0
Phenol red 15.0
Sodium pyruvate 110.0
Linoleic acid 0.084
Fumaric acid – 60.0
Æ-Ketoglutaric acid – 350.0
Malic acid – 600.0
Succinic acid – 60.0
Yeast extract – 2000.0
Culture media for animal cells 113
5.2 Main components of animal cell culture media
The culture medium must contain nutrients essential to the synthesis of
new cells and substrates for accomplishing cell metabolism, besides com-
pounds allowing physiological and catalytic functions, or which act as
cofactors. To maintain the conditions found in the original tissue from
which a particular cell originated, it is necessary for a culture medium to
contain: inorganic salts, sugars, amino acids, vitamins, lipids, organic acids,
proteins, hormones, carbon and nitrogen sources, micronutrients (organic
ions and minerals), and water as well as cell-specific substances. Some-
times, blood serum and antibiotics are also required, and in addition, the
culture must be kept free from inhibiting or toxic compounds.
Information on animal cell metabolism is essential for the formulation
of new culture media, as well as for the composition of the most
appropriate nutrient feed that may be used in various strategies of batch,
semi-batch or continuous culture that may be designed for the production
of the targeted protein. As already discussed in detail in Chapter 4 and
concisely presented in Figure 5.1, two fundamental substrates for animal
cells are glucose and glutamine.
The following sections will outline the major components of a typical
culture medium for animal cells as well as their typical concentrations and
their functions in cell metabolism.
5.2.1. Water
One of the basic components of a culture medium, and also one of the
most critical, is water. Animal cells are extremely sensitive to water
Glucose
Glucose-6-
phosphate
Pyruvate
Acetyl-CoA
Ribose-5-phosphate
Pentose
Cycle
Nucleic acids
Oxaloacetate
Malate
TCA
cycle
Fatty
acids
6-P-Gluconate
Glyceraldehyde
Dihydroxyacetone
Phospholipids
Glutamine
NH
4
⫹
NH
4
⫹
Glutamate
Alanine
α-Ketoglutarate
Lactate
Asparate
Protein
Nucleic
acid
Figure 5.1
Major pathways for central metabolism in animal cells (based on Shuler and Kargi, 2002).
114 Animal Cell Technology
quality, since this can be the source of contamination that can affect cell
growth. Potential contaminants include inorganic compounds (heavy
metals, iron, calcium, and chloride), organic compounds (detergents),
microbial-related contaminants (endotoxins and pyrogens), besides parti-
cles and colloids from several different origins. Since the presence of any
of these contaminants can prevent cell culture on any scale, strict purity
standards of water quality must be maintained. Efficient water purification
systems are, therefore, required, and can be based on multiple-distillation
systems or on equipment combining deionization, microfiltration, and
reverse osmosis. The water used in the preparation of industrial culture
media should be subjected to similar purification methods and should be
continuously monitored to ensure the physicochemical and microbio-
logical standards established by national pharmacopoeia. The standards
for water employed for pharmaceutical use in different countries are
described in Table 5.2, both for water for injection (WFI) and for purified
water (PW). While PW is used for the preparation of products not
requiring sterile or apyrogenic water, WFI meets the standards for PW as
well as for bacterial endotoxins, conductivity, and total organic carbon.
5.2.2 Glucose
Glucose is usually the main carbohydrate for animal cell growth, acting as
source of both carbon and energy (Be´rdad et al., 1993; Drews et al., 1995;
O
¨
hman et al., 1995; Mendonc¸a et al., 1999; Freshney, 2005). Normally,
this compound is added to the culture medium in concentrations varying
from 5 to 25 mM (0.9–4.5 g/L), but may be up to 56 mM (10 g/l).
Table 5.2 . Standards for the quality of purified water (PW) and water for injection (WFI)
(Slabicky, 199 4)
Parameter Units USA Great Britain Europe Japan
PW WFI PW WFI PW WFI PW WFI
Acidity/alkalinity mg.L
–1
1 2 1 2 0.1 0.1
Ammonium mg.L
–1
0.3 0.3 0.2 0.2 0.2 0.2 0.05 0.1
Bacterial
endotoxin
EU.L
–1
0.25 Pyrogen Pyrogen 0.25
Calcium mg.L
–1
0.5 0.5 1 1 1 1
Carbon dioxide mg.L
–1
44
Chlorides mg.L
–1
0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
Heavy metals (Pb) mg.L
–1
0.5 0.5 1 1 0.1 0.1 1
Magnesium mg.L
–1
0.6 0.6 0.6 0.6
Nitrates (N) mg.L
–1
0.2 0.2 0.2 0.2 0.2 0.2
Oxidizable
substances
mg.L
–1
5 5 1 0.5 5 10 5
pH 5–7 5–7
Sulfates mg.L
–1
0.5 0.5 0.5 0.5 0.5 0.5 0.5
Total organic
carbon
mg.L
–1
0.500
Total solids % 0.001 0.001 0.001 0.003 0.001 0.003 0.001 0.003
PW, purified water; WFI, water for injection; EU, endotoxin units; Pyrogen, pyrogen test (qualitative).
Culture media for animal cells 115
As detailed in Chapter 4, glucose is metabolized mainly through
glycolysis, forming pyruvate, which can be converted to lactate or acetyl
that can enter into the citric acid cycle, forming carbon dioxide and water.
Lactate accumulation in the culture medium indicates that the citric acid
cycle may not function in vitro similarly to the way it does in vivo. There
is evidence that a significant amount of carbon may be assimilated from
glutamine, and not from glucose, and that explains the large requirement
for this amino acid by some cell types. Insect cells, in general, accumulate
lactate at lower levels than mammalian cells. However, lactate can still be
produced by these cells in media with low oxygen concentrations.
The accumulation of lactate in high density cultures is a problem, since
it causes medium acidification. Also, this compound is toxic to the cells,
inhibiting cell growth when present in large concentrations. The substitu-
tion of part of the glucose by mannose, fructose, galactose, or maltose
reduces glucose consumption rate and, as a consequence, also reduces the
lactate formation rate. However, total substitution may affect protein
glycosylation patterns, altering proteins such as antibodies.
5.2.3 Amino acids
Essential amino acids are those not synthesized by the organism and are
required by animal cells in culture. These include specific amino acids such
as cysteine and tyrosine but the requirement varies between cell lines.
Amino acids are necessary for protein, nucleotide, and lipid synthesis and,
in addition, may be used as an energy source.
These compounds can be provided as a defined mixture or in the form
of protein hydrolysates, such as lactoalbumin, or plant-derived hydroly-
sates, like colza (Deparis et al., 2003), soybean (Donaldson and Shuler,
1998; Heidemann et al., 2000; Ikonomou et al., 2001), wheat (Heidemann
et al., 2000; Ikonomou et al., 2001; Ballez et al., 2004), and rice (Heide-
mann et al., 2000; Ikonomou et al., 2001; Ballez et al., 2004). Supplementa-
tion with yeast extract also provides additional amino acids.
Traditionally, from 0.1 to 1 mM of each amino acid is added to the
culture medium, including both essential and non-essential amino acids.
The concentrations of amino acids added to insect cell culture media are
much higher than those found in media for vertebrate cells (Echalier,
1997), probably due to the fact that higher amino acid concentrations are
found in insect hemolymph (insect body fluid) in comparison with blood
serum.
Glutamine, methionine, and serine are growth-limiting amino acids
(Freshney, 1992). Glutamine acts as a source of carbon, nitrogen, and
energy, and is normally added to the culture medium at high concentra-
tions, varying from 1 to 5 mM. Special attention should be given to its
stability, since glutamine spontaneously degrades by cyclization to pyrro-
lidone carboxylic acid in the culture medium. Glutamine is prone to
thermal degradation (at 358C, for instance, glutamine is 50% degraded in
only 9 days), and therefore, should be periodically replenished when
required. The main metabolite generated from glutamine metabolism is
ammonium which, similarly to lactate, presents toxic effects and is a cell
growth inhibitor when accumulated in large quantities. It can be converted
116 Animal Cell Technology
enzymatically to glutamic acid, leucine, and isoleucine by enzymes found
in cells and also in the blood serum normally added to culture media
(Freshney, 1992).
5.2.4 Vitamins
Many cells require media supplemented with complex B vitamins, while
other vitamins are presumably supplied by the addition of serum to
culture media. Nevertheless, when serum-free media are employed, not
only the water-soluble vitamins should be provided, but also the lipid-
soluble ones, such as biotin, folic acid, niacin, panthotenic acid, thiamine,
and ascorbic acid, as well as the vitamins B
12
, A, D, E, and K.
Vitamins are used in very low amounts as enzyme cofactors, essential
for general cell metabolism. While ascorbic acid acts in collagen synthesis,
vitamin A affects cell growth and differentiation. Vitamin K is required for
gamma-carboxylation and for correct protein processing. Vitamin E is an
antioxidant agent, whereas vitamin D regulates calcium ion transport and
acts as a hormone, being toxic when supplied in excess.
Besides serum, yeast extract can also be used as an effective vitamin
supply to culture media.
5.2.5 Salts
The salts most commonly added to the culture medium are Na
þ
,K
þ
,
Mg
2þ
,Ca
2þ
,Cl
–
,SO
4
2–
,PO
4
3–
, and HCO
3
–
. These ions are important in
the maintenance of the ionic balance and osmotic pressure, besides acting
as enzymatic cofactors. Salts are the components that contribute most to
the increase in culture medium osmolality. Na
þ
,K
þ
, and Cl
–
have relevant
action in the regulation of the cellular membrane potential, while SO
4
2–
,
PO
4
3–
, and HCO
3
–
are important for macromolecular synthesis, as well as
regulators of intracellular charge (Freshney, 2005). In addition, HCO
3
–
acts in the binding of iron to transferrin and in culture medium buffering.
For suspension cultures, the concentration of calcium and magnesium
should be kept low to prevent cell aggregation and adhesion. Other metals,
such as iron, manganese, selenium, vanadium, zinc, copper, and molybde-
num, are usually added to the culture medium, but at reduced concentra-
tions, and mainly if the medium is not supplemented with animal serum.
5.2.6 Serum
Blood serum, usually bovine-derived (calf or fetal bovine), contains amino
acids, growth factors, vitamins, proteins, hormones, lipids, and minerals,
among other components, as indicated in Table 5.3. Besides fetal bovine
serum, serum from horse (equine), and even from humans (less common)
can also be used. The main functions of serum are to stimulate growth and
other cellular activities through hormones and growth factors, to increase
cellular adhesion through specific proteins, and to supply proteins for the
transport of hormones, minerals, and lipids (Freshney, 2005). Supplemen-
tation with bovine fetal serum is performed at concentrations from 2 to
20% in volume.
Culture media for animal cells 117
In the case of insect cells, insect hemolymph can be used as an
alternative to bovine serum. However, the availability of hemolymph is
much more restricted.
5.2.7 Other components necessary for cell culture
Besides the compounds already mentioned, some adherent cell lines
need proteins of the extracellular matrix (ECM) for efficient adherence
to the support and for cell growth. Many ECM proteins, such as
fibronectin, are present in serum, as shown in Table 5.3, while others,
such as collagen, are secreted by cells constitutively or after their
stimulation with a growth factor. Another ECM protein, laminin, can
Table 5.3 Serum components and their functions in the animal cells cultured in vitro (adapted
from Freshney, 2005)
Component Probable function
Proteins
Albumin Osmotic control and buffering agent, transport of
lipids, hormones and minerals, mechanical
protection
Fetuin Cell attachment
Fibronectin Cell attachment
Æ
2
-Macroglobulin Trypsin inhibitor
Æ
1
-Antitrypsin Trypsin inhibitor
Transferrin Binds iron
Growth factors
Endothelial growth factor (ECGF) Mitogen
Epidermal growth factor (EGF) Mitogen
Fibroblast growth factor (FGF) Mitogen
Insulin-like growth factors (IGF-1 and IGF-2) Mitogen
Platelet-derived growth factor (PDGF) Mitogen and major growth factor
Interleukin-1 (IL-1) Induces interleukin-2 release
Interleukin-6 (IL-6) Promotes differentiation
Hormones
Hydrocortisone Promotes attachment and proliferation
Insulin Promotes glucose and amino acids uptake
Growth hormones Mitogen (present in fetal serum)
Metabolites and nutrients
Amino acids Cellular proliferation
Glucose Cellular proliferation
Keto-acids (e.g. pyruvate) Cellular proliferation
Lipids (e.g. cholesterol) Membrane synthesis
Minerals
Iron, copper, zinc, selenium Enzyme cofactors and constituents
Inhibitors
ª-Globulin
Bacterial toxins from prior contaminants
Chalones (tissue-specific inhibitors)
118 Animal Cell Technology
be used as an alternative to fibronectin, especially for epithelial cell
cultures. Growth factors, present in very low amounts, are usually small
peptides (5–30 kDa). Among them, the following types are prominent:
FGF (fibroblast growth factor), EGF (epidermal growth factor), NGF
(nerve growth factor), TGF (transforming growth factor), PDGF (plate-
let-derived growth factor), the insulin homologous IGF-1 and IGF-2,
and interleukins (Jenkins, 1991).
According to Jenkins (1991), the mitogenic factor FGF is isolated from
bovine brain in two forms, acid FGF (pI 5.6) and basic FGF (pI . 9.0).
These factors bind strongly to heparin. Basic FGF (bFGF) presents
mitogenic action through calcium influx and activation of quinase C
proteins, being a potent mitogenic agent for several types of cultures
derived from mesodermal and neuroectodermal cells, as well as for
several transformed cell lines. This compound also promotes differentia-
tion of adipocytes and ovary granulous cells, inducing the synthesis of
ECM proteins such as collagen type IV. bFGF has been used to enhance
the survival of new cell lines derived from primary cultures and also to
eliminate the use of feeder cells during hybridoma cloning. Feeder cells
(such as irradiated fibroblasts or cells from peritoneal exudates) are used
to condition culture media with an undefined mixture of growth-promot-
ing substances, particularly when hybridomas are present at low densi-
ties.
EGF can be isolated from mouse submaxillary glands and also from
human urine. It is a potent mitogenic agent for many primary cultures and
for mesenchymal and epithelial cells, having a synergistic effect with other
growth factors, such as IGF-1 and TGF.
NGF is another factor purified from mouse submaxillary glands, and it
can also be obtained from bovine brain and placenta. NGF is not a potent
mitogenic agent, but it induces differentiation and increases the survival of
sympathetic neurons and PC12 cells in culture.
The factors TGFÆ and TGF were initially observed in fibroblast
cultures of mouse kidney cells transformed by retrovirus (Sporn et al.,
1987). TGFÆ induces cellular growth independently of adhesion, and its
action is similar to that of EGF (Derynck, 1988). However, several cell
lines that produce endogenous TGFÆ, like A431, can have their growth
inhibited by EGF. TGFÆ is found naturally in embryonic kidney, adult
brain, pituitary gland, skin, and in the placenta.
TGF is a factor commonly purified from human platelets, which
predominantly secrete TGF-1 (Jenkins, 1991). The biologically inactive
TGF precursor (220–235 kDa) circulates in the blood as a protein and
possible protease carrier. Most of the mammalian cells in culture secrete
small amounts of TGF precursor and have TGF receptors. TGF
promotes growth in several lineages of mesenchymal cells when in
combination with TGFÆ or EGF. In contrast, isolated TGF inhibits the
growth of many cell lines in monolayer culture, which could result from
its capacity to stimulate the secretion of ECM proteins (collagen, fibronec-
tin, and glucosaminoglycans) and protease inhibitors (Moses et al., 1987).
The inhibition of monolayer growth by TGF is more prominent in
epithelial cells, endothelial cells (antagonized by the bFGF), stem cells,
and lymphocytes.
Culture media for animal cells 119
PDGF is one of the most common serum components (40–60 ng/ml). It
is also secreted by endothelial and placenta cells, and by several cell lines
such as BHK and 3T3-fibroblasts. PDGF has synergetic action with EGF
and IGF-1 and is a potent mitogen.
Another fundamental compound for cellular growth is insulin, which
presents weak affinity to IGF-1 receptors. Insulin can activate several
mitogenic responses, through IGF-1 receptors when in large doses. How-
ever, insulin is also added to most serum-free media due to its ability to
promote anabolic metabolism, such as oxidation processes, glycogen
synthesis, and amino acids transport (Jenkins, 1991).
Peptides homologous to insulin can also act as growth factors, such as
somatomedin-C (Daughaday and Rotwein, 1989). IGF-1, secreted in small
amounts by many tissues, acts on several mesenchymal cells, while IGF-2,
produced by fetal liver, muscle, skin, and adult brain, is known for its
growth stimulatory activity.
Some cells, such as hybridomas, stem, and hematopoietic cells may
require a class of proteins known as interleukins, especially IL-6. This
compound can substitute for feeder cells (such as those of the peritoneal
exudates or spleen, fibroblasts or timocytes) in the post-fusion stage of
hybridoma cultures. IL-6 is secreted by monocytes, T lymphocytes, and
endothelial cells and is effective in the stimulation of hybridomas of
different species, including lineages that are difficult to cultivate, such as
human–mouse and rat–mouse hybrids. IL-6 also presents a synergistic
effect with other interleukins to increase antibody production.
Some growth factors act as carrier proteins in the culture medium. These
proteins can serve as carriers in lipid transport (as in the case of the
albumin) or for metal ion transfer to cells (such as transferrin). Albumin
can act as a buffering or a detoxifying agent (due to its binding to
endotoxin and pyrogens), as well as serving as a carrier for vitamins,
hormones, or other micronutrients. Transferrin, on the other hand, is a
protein with a large capacity as an iron carrier. Many mammalian cells
possess transferrin-specific receptors which capture transferrin/Fe
3þ
com-
plexes.
Lipids also can be beneficial for cells in culture, since some substances
absorbed by the cells need to be solubilized in lipids, or in some cases the
toxicity of compounds may be reduced by complexation with lipids. The
absence of essential lipids such as linoleic acid, lecithin, cholesterol,
ethanolamine, or phosphorylcholine can result in the decrease of cloning
efficiency and in reduction in the size of colonies, as shown for insect cells
by Echalier (1997). However, one of the difficulties in supplying lipids at
reasonable concentrations is their low solubility. To circumvent this
limitation, lipids can be emulsified with complexing agents such as
Pluronic
1
F68 or cyclodextrin (Maiorella et al., 1998).
Antibiotics such as penicillin, streptomycin, and amphotericin B can
also be added to the culture medium to prevent microbial growth. How-
ever, their use should be kept to a minimum level, because some antibiotics
can have cytotoxic effect, even when high serum concentrations are used.
Besides, the continuous use of antibiotics can result in the selection of
resistant microorganisms, inducing a chronic contamination of the culture
(Freshney, 1992).
120 Animal Cell Technology
5.3 Advantages and limitations of the use of media
supplemented with animal serum
As already mentioned, the most frequently employed serum in animal cell
culture is bovine fetal serum; however, sera of calves, horses, and even
human sera are also used. Proteins of the human plasma are obtained in
substantial amounts as a result of routine therapeutic production of blood
products. In these conditions, large amounts of albumin, transferrin and
insulin, as well as growth factors, can be obtained. The fractions with no
therapeutic application are usually discarded, but they can be processed
with the purpose of supplementing low protein media, as a direct substi-
tute for bovine fetal serum, allowing the growth of a wide range of cells.
To use these proteins, cell adaptation to the medium containing the new
products is necessary, and this process can take several weeks. In addition,
limitations such as the possibility of introduction of human pathogens in
the culture should be considered.
Fetal bovine serum is the favorite because it presents low immuno-
globulin and high growth factor concentrations. Its main advantage is that
it allows the same medium to culture different cell lines. For production,
the blood of bovine fetuses is collected by heart puncture and, after slow
coagulation, it is centrifuged, resulting in the serum in the supernatant.
The entire process should be performed at low temperatures, and the
animals should be free from infectious diseases, such as rinderpest, foot-
and-mouth (aftosa), and bovine spongiform encephalopathy (‘‘mad cow
disease’’). Fetal bovine serum is scarce since, on average only 7–9% of
slaughtered animals are estimated to be pregnant and each fetus supplies
from 0.2 to 0.5 l of blood with only half the volume becoming serum.
Thus, around 44–144 animals have to be slaughtered to obtain 1 l of fetal
bovine serum. Beside the low process yield, there is also an ethical aspect.
The animals can suffer if at the moment of blood collection their lungs are
insufflated with air, increasing the oxygen blood concentration to levels
compatible with animal consciousness (Van der Valk et al., 2004).
In addition to the factors that increase cellular adhesion and the nutri-
tional factors, serum also presents culture-stimulating (growth factors,
hormones and proteins) and protecting agents, both for biological protec-
tion (antitoxin, antioxidant, antiprotease) and for prevention of mechanical
damage. However, fetal serum is one of the most expensive components in
culture medium. In addition, serum supplementation can significantly
increase the complexity and the cost of the targeted product recovery and
purification process due to the presence of many proteins and growth
factors, among other components, most of them undefined. Bovine fetal
serum can contain more than 1000 different components.
Another factor to be taken into account when using animal serum is the
potential risk for human health, due to the possible presence of adventi-
tious agents, such as virus and proteins as prions. In addition, serum can
contain contaminants such as bacteria, fungi, and mycoplasmas (small
bacteria without cellular walls), which can negatively affect cell culture.
Another strong limitation to the use of bovine fetal serum is its
variability between different lots and suppliers, which hinders the standar-
dization of the culture medium and the reproducibility of culture perform-
Culture media for animal cells 121