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1.  Introduction Cell
culture is a technique that is used to grow and maintain cell types in a
laboratory. The role of medium in cell culture is to provide all the
requirements that cells would normally obtain for growth in vivo. Serum is most commonly used as a supplement to cell
culture media, providing carrier proteins, attachment and spreading factors,
growth factors, hormones and nutrients(1). When
animal cell culture was in its early stages, it was found that a small amount
of serum could support the growth and proliferation of cells. Other fluids were
experimented with, such as bovine colostrum and amniotic fluid but serum was
the most efficient, with foetal bovine serum (FBS) being the most widely used
serum(2)(3).  The use
of FBS in human and animal cell culture media is still common practice. It is
obtained from bovine foetuses removed from pregnant cows during slaughter,
where harvesting is most commonly through a cardiac puncture without
anaesthesia(4). Suffering can only occur if they
inflate their lungs with air and their blood oxygen is increased to a level
compatible with awareness(5). Aside from animal welfare
concerns, of the potential suffering during harvesting, there are problems, with
the use of FBS, in terms of quality and reproducibility of in vitro results(6). A major disadvantage in using
serum is the wide possibility of contaminants. Protein concentration is one of
the main contaminants, with it being reported that if a cells native protein
has the same function as a sera protein, it is possible the two cannot be
separated. This results in scientists being restricted in their research when
looking for mechanisms of a specific protein(7). With the
many concerns that surround the use of FBS, several strategies have been
developed to replace it FBS in cell culture media in terms of the 3Rs,
Refinement, Reduction and Replacement(8). Over the years, several
alternatives to serum-based media have been studied, as shown in table 1.
Serum-free media does not contain any serum or plasma and instead are
supplemented by essential components. Xeno-free is media containing only
human-derived supplements in both the cell culture and the reagents added,
whereas animal component free is media that is not exposed or derived from any
animal or human during manufacturing(9,10). This
paper reviews the advantages and disadvantages of foetal bovine serum and
serum-free media as an alternative, including the essential components that
must be supplemented.                Table 1. Classification of cell culture media
and their definitions.


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· Media containing animal or human
· Additional growth factors are
sometimes supplemented.

· Human AB serum


· Media containing neither serum
nor unprocessed plasma but may contain discrete proteins or bulk protein
· In replacement of serum or
plasma, supplements are sometimes included.

· Animal/human-derived supplements
and hydrolysates
· A mixture of
human/animal-derived or recombinant proteins, hormones, growth factors and


· Media containing only
human-derived supplements, so that the finished product is considered to be
human sourced.
· The term xeno-free denotes both
primary and secondary levels, therefore the cell culture and all reagents
used should be xeno-free themselves.

· Human serum or plasma
· Human-derived supplements
· Plant hydrolysates
· Mixture of human-derived of
recombinant proteins, hormones, growth factors and lipids


· Media containing neither animal
nor human-derived components.
· The final product is not
derived, manufactured with or exposed to any animal or human-derived
components during manufacturing operations.

· Plant hydrolysates
· A mixture of recombinant
materials that are expressed and processed from qualified cell lines


· Media containing no proteins,
growth factors, hydrolysates or components of unknown composition.

· Recombinant materials such as
proteins, hormones, cytokines and growth factors


· Media containing no proteins but
often containing hydrolysed proteins and small peptides like insulin with low
molecular weight.
· Protein-free media may contain
ill-defined components such as lipids and therefore may not be chemically

· Hydrolysed proteins
· Small peptides like insulin

  2.  Foetal Bovine Serum  2.1      The composition of FBS FBS
is very hard to define as it comprised of a complex mixture of components,
including biomolecules with different physiologically balanced growth-promoting and growth-inhibiting
activities. Rauch et al. outline the major functions of serum to be providing hormonal
factors, stimulating cell growth and proliferation, to promoting differentiated
functions, and providing transport proteins, minerals, trace elements, lipids,
attachment and spreading factors, and stabilizing and detoxifying factors needed
for maintaining pH(7). What makes FBS superior to serum from adult
animals is its low gamma-globulin content, as if antibodies levels are high it
may inhibit growth and proliferation.2.2      Advantages of FBS FBS has
many advantages, which is why, in cell cultures, it is the preferred animal
serum. It is rich in proteins, enzymes, growth factors and other components.
The foetal growth factors and hormones are what stimulate the cells to
proliferate. FBS also regulates cell membrane permeability, so acts as a
carrier for enzymes, lipids and micronutrients into the cell. In addition, it
serves as a buffer to the cell culture system against disruptions and toxic
effects like endotoxin, pH change and proteolytic activity(11). ***Additionally,
FBS is abundantly available due to being a by-product of pregnant cows that go
to slaughter and is therefore commercially available (12). Lastly, the use of a serum-supplemented
media, like FBS, reduces time and effort spent on developing specific media for
each cell type and FBS is suitable for a range of different cell types(1).  2.3      Disadvantages of FBS 2.3.1    Ethical During
the procedure of harvesting, a bovine foetus will experience anoxia, an acute
lack of oxygen. This is due to the blood supply from the placenta stops upon
the death of the mother(4). There have been studies on the resistance
of mammal foetuses to anoxia in comparison to adults of the same species. Using
rabbits as an example, adult rabbits can survive for 1.5 minutes in pure
gaseous nitrogen, whereas rabbit foetuses at 29 day gestation survive for an
average of 44 minutes(13). If anoxia is upheld over the
time of last gasp, permanent brain damage occurs, as shown in dogs, rhesus
monkeys and guinea-pigs(13).  However, one
of the safeguards, to prevent suffering of the foetus, in a report by van der
Valk et al. states that the procedure of foetal blood collection must not begin
until at least 5 minutes after an effective neck cut has been completed and
that the foetus must remain severely anoxic throughout the procedure(14,15). This contradicts a study from
only two years before by Jochems et al. as they state that discomfort might be
experienced until actual brain damage occurs(4). So, where van der Valk believes
that the foetus will not experience suffering during anoxia, Jochems deems that
suffering could be experienced during this time until brain damage(4,14). Another safeguard listed in the
van der Valk report states that if the foetus takes a breath then, to avoid
suffering, it must be stunned with a captive bolt. Conversely, in the Jochems
et al. article they say that harvesting without refinement of pain avoidance,
such as stunning with a captive bolt can be considered immoral(4,14). This statement by Jochems
clearly states that stunning with a captive bolt would be immoral, yet van der
Valk believes that the same action should be used to prevent suffering. Nonetheless,
in an article in 2003, by David Mellor, the chairman of the National Animal Welfare
advisory committee, they support the three safeguards that van der Valk states(15). The guidelines from Mellor can
be seen in Table 2 below.      Table 2.
The Guidelines for the humane slaughter of bovine foetuses as set out by the
National Animal Welfare advisory committee(15).



Slaughter of the pregnant dam must be humane

ensure the dam remains insensible to pain and distress, the slaughter must
meet high welfare standards.

Shortage of oxygen in the foetal brain prevents foetal suffering

If the
foetus doesn’t inflate its lungs, it cannot become conscious and therefore
cannot suffer.

Where practical, leave the foetus in the uterus until it is dead

foetuses should be left inside the unopened uterus until they are dead as
foetal death or irreversible brain damage prevents suffering of the foetus.

4. The
earliest removal time is five minutes after the maternal neck or chest cut

ensure that brain electrical activity is flat, foetuses must remain in the
uterus for a minimum of five minutes.

5. Lung
inflation must be prevented if a living foetus is exposed to air

If a
foetus is removed from the uterus, either its head must remain inside the
amniotic sac or its windpipe be clamped to ensure it remains unaware.

Foetuses exposed alive may be killed immediately

exposed, the foetus can be killed by a neck cut or by captive bolt
destruction of the brain.

Unaware (unconscious) foetuses die without suffering

the foetus does not inflate their lungs, they cannot become aware and therefore
cannot suffer.

     2.3.2    Scientific Batch-to-batch variation and fraudulent
marketing However,
one of the main disadvantages to FBS is that batch-to-batch variation is unavoidable.
This means that before purchase, each batch must be tested as multiple
different growth factors or growth inhibition factors could be present. Ultimately,
variation in the concentrations of the components can leady to experimental
variability and as a result limit the inter-laboratory reproducibility of an
experiment(8). In
2013, GE Healthcare issues a product information to customers which stated that
batches of FBS that had been produced by PAA Laboratories between 2008 and 2013
could be subject to “label non-conformances”(6). The statement released was: “These products may contain added
adult bovine serum albumin (BSA) of United States origin, water, and/or cell
growth promoting additives”. This warning from GE Healthcare about the purity
of FBS prompts the question of how well the FBS market is regulated. The US
Food and Drug Administration (FDA) reported that 143 batched of FBS were
affected by this incidence(16). This incidence may have a
substantial impact on thousands of cell culture experiments.Another
incidence of abuse to the regulations was in 1994 in New Zealand. It was
reported that 30,000 litres of FBS from New Zealand was sold worldwide.
However, only 15,000 litres of high-quality FBS was collected from New Zealand
in that year. Even in 2014, the exact figures for the production rate of FBS
throughout the world was unavailable(6). This raises major suspicions,
as there is still a possibility that FBS is being blended with other sera to
keep up with the rising demand from the industry. ContaminantsAnother
major disadvantage is that the serum can be contaminated with bacteria,
mycoplasmas, fungi, viruses, yeast, endotoxins and immunoglobulins(4). In a study done by Bieback et
al. in 2009 found that in four FBS-supplemented cultures, three showed
bacterial contamination, meaning it was necessary to discard three of said
cultures(17).   3.  Serum-Free Media 3.1       Introduction Serum in
media introduces unknown variables into the cell and tissue culturing, so one
of the main reasons serum-free media (SFM) was introduced is because the
components can be defined. SFM can also be cell-specific. There have been
studies by Barnes and Sato, 1980; Taub, 1990 and Bjare, 1992 investigating various
combinations of hormones, nutrients and purified proteins to replace serum in
specific cell lines and types(3,18–20). They found that the combinations
were unique to each cell type and therefore in many cases it cannot supplement
the growth of other cell types. The high specificity, that derives from knowing
the composition of the media, gives the opportunity for specific stimulation
and differentiation for individual cell types. However, general-purpose serum-free media has not been developed and is likely to be an unachievable
goal.  3.2       What serum-free media requires To
develop serum-free media, it is important to understand its function in cell
culture. In addition to supplying hormones for growth, serum also provides
proteins that bind to vitamins, lipids, metals and hormones(18). When serum is absent, substitute
components must be added to replace the major function in its place. The main
components required for a serum-free media are hormones, growth factors,
cell-attachment factors and transport binding proteins(14,20). 3.2.1    Serum
albumin In
serum-free preparations, transport proteins are required to carry hormones,
minerals and trace elements and bovine serum albumin is the most commonly used.
It can also act as protection from shear stress as it can bind to toxic
components in the culture medium. Xeno-free formulations typically use human
serum albumin (HSA), however the performance of it can depend on the source as
there are varying methods of isolation from the blood, which in turn may affect
the characteristics of the product. Therefore, to obtain the optimal product
for a specific cell culture, screening of HSA suppliers is needed. To avoid
using albumin, ACF medium preparations can add fatty acids, lipids,
phospholipids and trace elements as a replacement(10,19).  3.2.2    Insulin A
clinical grade recombinant insulin is most widely used in serum-free formulations
in order to uptake glucose, metabolise lipids and synthesise DNA. It is usually
in the concentration range of 2-10 mg/L. An alternative to insulin is
recombinant IGF-1 in XF or ACF. Due to the direct activation of the IGF-1
receptor, it is used when yielding an enhanced performance with specific cell
types(3,10).  3.2.3    Human
transferrin Human
transferrin is a carrier protein. Its function is to transport iron into the
cell to optimize cell growth and proliferation. Derived from human plasma, it
is collected as source plasma and approved for human use. Recombinant
equivalents have limited availability commercially at a higher price with
comparable performance(3). Alternatively, salts such as
iron ethylenediaminetetraacetic acid and other iron chelators can be used in
ACF media. However, there’s a chance that the salts could have negative results
on cell growth, due to free radicals forming and the lack of iron available to
the cells(10,15).  3.2.4    Hormones Glucocorticoids,
thyroid hormones and oestrogens can be used in serum-free media and are the
most commonly used. An increase in proliferation occurs in adherent cells when
adding hydrocortisone, progesterone and dexamethasone. Specific combinations of
hormones added to ACF media can be a key component, especially when certain
cell types are used in cell therapy applications(10,15).  3.2.5    Growth
factors Growth
factors stimulate cell proliferation and maintain the characteristics of a
cell. XF and ACF media use basic fibroblast factor, epidermal growth factor,
transforming growth factor beta, vascular endothelial growth factor and
platelet-derived growth factor(3). Available as recombinant
proteins, they are widely used for cell therapy. The key to achieving
optimised, cell specific, serum-free ACF medium are specific growth factors,
concentrations and synergistic effects. At premium pricing, cGMP growth factors
are manufactured, but are used less often(10,22).  3.3       Serum-free investigations of specific
cell types 3.3.1    Epithelial
cells In the
past ten years, epithelial cells have been predominantly cultured in defined
media due to the testing of hormonal supplements. For epithelial cells to grow,
they require the supplementation of hormones in the media. Tsao et al.
investigated colony formation of human epidermal keratinocytes in rich media
supplemented with hormones. The medium also contained epidermal growth factor,
hydrocortisone, insulin, transferrin, progesterone, ethanolamine and
phosphoethanolamine. The medium was high in calcium, which prompted the cells
to grow less but differentiate more. Although a mixed inoculum of fibroblasts,
keratinocytes and epithelial cells was used, the medium was specifically
selective towards the epithelial cells(21). Another study was conducted by
Lechner et al. using the same supplements as Tsao, except for progesterone. The
study proved that normal human bronchial epithelium cells could be cultivated
in dishes coated with collagen, albumin and fibronectin(22). It has
been discovered that epithelial cells grown in
vitro will often be expressed differently than in vivo(19). Kirk and Alvarez, like Tsao,
cultured epithelial cells in a defined medium supplemented by hormones. The study
goes on to discover that the cells would respond to hormones and retain their
structure for several months in the culture, due to the formation of vesicular
structures(23). In contrast, a study by
Reznikoff et al. demonstrated that epithelial cells grown in Ham’s F12 medium,
didn’t improve the growth when compared to media containing serum. This was
proved by the mitogenic reaction when serum was added and an increase in
differentiation of the cells when the concentration of calcium was increased(24).  4.  Serum Substitutes A study
by Fang et al. investigated the growth capability of five neck and head
squamous carcinoma cells (OECM-1, TW01, HONE-1, SCC25 and FaDu) and one
dysplastic oral keratinocyte cell (DOK) lines in six bovine calf serum based cell
cultures in comparison to FBS. They were cultured in the FBS alternatives for
30 serial passages to determine their ability to support long term growth. The
methods used were assessing the morphology of the cells in each culture, plating
efficiency assay and multiple functional assays.  The sera
used in addition to FBS were newborn calf serum, bovine calf serum (CS),
iron-supplemented calf serum(ICS). Three bovine serum-based alternatives that were
used fetalgro bovine growth serum(FG), cosmic calf serum(CCS) and foetal clone three
serum(FC3).  From
their results, newborn calf serum containing medium was could not sufficiently
support the proliferation of the cell lines and therefore were not able to
support long term growth. In contrast, many vendors actually recommend newborn
calf serum as a cheaper replacement for FBS. A previous study discovered that
certain sera may have reduced cell detachment due to low trypsin inhibitor
activity, but a duplicate study including Accutase (a detachment reagent) still
exhibited poor extension and delayed attachment in newborn calf serum.  In the
TW01 cell line, a nasopharyngeal carcinoma cell line, FC3, CCS and FG showed
growth rates similar to FBS, but CS and ICS had a reduced proliferation and
were more compact than cells grown in FBS. CCS and FG showed slight granularity
of the cytosol and the morphology in FC3 appeared similar to that of FBS(25).  In
contrast, the HONE-1 nasopharyngeal carcinoma cell line cultured in FC3 almost
better than FBS. CCS and FG showed a reduction in growth in the first ten
passages but in the following tests they grew similarly to FBS. The cells
cultured in CS and ICS again showed reduced proliferation but instead showed
increased granularity. Whereas FG and CCS exhibited as more compacted growth of
HONE-1 cells(25).  The proliferation
in the two cell lines TW01 and HONE-1, which are both nasopharyngeal carcinoma
cell lines, were similar for FC3. However, in TW01 cells, ICS and CS were
compact with CCS and FG showing granularity, but in HONE-1 cells, the cells
were showing granularity for ICS and CS and compact in CCS and FG(25).    5. Conclusion The future of cell culturing requires more
in depth investigation into alternatives to foetal bovine serum but of the many
types of culture supplements, each have their advantages and disadvantages.
This paper has reviewed how
foetal bovine serum works, along with the advantages and disadvantages to using
it for cell culture, and the alternatives available.  Foetal
bovine serum does have its advantages as a supplement to cell culture media,
however, from this paper, the severity of some of the disadvantages outweigh
the advantages. While the worldwide production rate of FBS is unavailable,
there will always be an uncertainty to composition of FBS being sold on the
market. If the FBS market was regulated thoroughly, the industry could buy FBS
in confidence that it is of high-quality.
Due to the shortage of FBS, several alternatives have been developed. Sera from
other animals, like goats or horses, have been suggested as potential
alternatives, but as a result of them only supporting the growth of a small
amount of cell lines, their submissions were limited.

Human serum and human platelet lysates were also reported to be a viable FBS alternative.
As they act as xeno-free media, their major advantage as human serum-derived
supplements is that they are non-xenogeneic when used with human cell lines. However, they are strictly used to culture
human cell lines only for therapeutic purposes, such as stem cells and
mesenchymal stromal cells, due to the limited availability.

Panexin is a chemically defined serum replacement for
the cultivation of cells under serum-free culture conditions or to
significantly reduce the amount of serum used in cell culture. It supports the
growth of many cell types in an optimum manner without any extra handling when
compared to serum. In addition, as Panexin is fully chemically defined, no lot
testing is required. It contains no growth factors, which means a defined
proliferation can be seen(26).

As a result, there is a need for research into whether
Panexin is a suitable replacement for foetal bovine serum.


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