1. plasma and instead are supplemented by essential components.

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Last updated: September 27, 2019

1.  Introduction Cellculture is a technique that is used to grow and maintain cell types in alaboratory. The role of medium in cell culture is to provide all therequirements that cells would normally obtain for growth in vivo. Serum is most commonly used as a supplement to cellculture media, providing carrier proteins, attachment and spreading factors,growth factors, hormones and nutrients(1). Whenanimal cell culture was in its early stages, it was found that a small amountof serum could support the growth and proliferation of cells. Other fluids wereexperimented with, such as bovine colostrum and amniotic fluid but serum wasthe most efficient, with foetal bovine serum (FBS) being the most widely usedserum(2)(3).  The useof FBS in human and animal cell culture media is still common practice.

It isobtained from bovine foetuses removed from pregnant cows during slaughter,where harvesting is most commonly through a cardiac puncture withoutanaesthesia(4). Suffering can only occur if theyinflate their lungs with air and their blood oxygen is increased to a levelcompatible with awareness(5). Aside from animal welfareconcerns, of the potential suffering during harvesting, there are problems, withthe use of FBS, in terms of quality and reproducibility of in vitro results(6). A major disadvantage in usingserum is the wide possibility of contaminants. Protein concentration is one ofthe main contaminants, with it being reported that if a cells native proteinhas the same function as a sera protein, it is possible the two cannot beseparated. This results in scientists being restricted in their research whenlooking for mechanisms of a specific protein(7). With themany concerns that surround the use of FBS, several strategies have beendeveloped to replace it FBS in cell culture media in terms of the 3Rs,Refinement, Reduction and Replacement(8).

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Over the years, severalalternatives to serum-based media have been studied, as shown in table 1.Serum-free media does not contain any serum or plasma and instead aresupplemented by essential components. Xeno-free is media containing onlyhuman-derived supplements in both the cell culture and the reagents added,whereas animal component free is media that is not exposed or derived from anyanimal or human during manufacturing(9,10). Thispaper reviews the advantages and disadvantages of foetal bovine serum andserum-free media as an alternative, including the essential components thatmust be supplemented.

                Table 1. Classification of cell culture mediaand their definitions. Term Definition Supplement Example Serum-based · Media containing animal or human serum. · Additional growth factors are sometimes supplemented. · FBS · Human AB serum Serum-free · Media containing neither serum nor unprocessed plasma but may contain discrete proteins or bulk protein fractions. · 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 lipids.

Xeno-free · 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 Animal component-free · 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 Chemically defined · Media containing no proteins, growth factors, hydrolysates or components of unknown composition. · Recombinant materials such as proteins, hormones, cytokines and growth factors Protein-free · 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 defined.

· Hydrolysed proteins · Small peptides like insulin   2.  Foetal Bovine Serum  2.1      The composition of FBS FBSis very hard to define as it comprised of a complex mixture of components,including biomolecules with different physiologically balanced growth-promoting and growth-inhibitingactivities. Rauch et al. outline the major functions of serum to be providing hormonalfactors, stimulating cell growth and proliferation, to promoting differentiatedfunctions, and providing transport proteins, minerals, trace elements, lipids,attachment and spreading factors, and stabilizing and detoxifying factors neededfor maintaining pH(7).

What makes FBS superior to serum from adultanimals is its low gamma-globulin content, as if antibodies levels are high itmay inhibit growth and proliferation.2.2      Advantages of FBS FBS hasmany advantages, which is why, in cell cultures, it is the preferred animalserum. It is rich in proteins, enzymes, growth factors and other components.The foetal growth factors and hormones are what stimulate the cells toproliferate. FBS also regulates cell membrane permeability, so acts as acarrier for enzymes, lipids and micronutrients into the cell. In addition, itserves as a buffer to the cell culture system against disruptions and toxiceffects like endotoxin, pH change and proteolytic activity(11).

 ***Additionally,FBS is abundantly available due to being a by-product of pregnant cows that goto slaughter and is therefore commercially available (12). Lastly, the use of a serum-supplementedmedia, like FBS, reduces time and effort spent on developing specific media foreach cell type and FBS is suitable for a range of different cell types(1).  2.3      Disadvantages of FBS 2.3.

1    Ethical Duringthe procedure of harvesting, a bovine foetus will experience anoxia, an acutelack of oxygen. This is due to the blood supply from the placenta stops uponthe death of the mother(4). There have been studies on the resistanceof mammal foetuses to anoxia in comparison to adults of the same species. Usingrabbits as an example, adult rabbits can survive for 1.

5 minutes in puregaseous nitrogen, whereas rabbit foetuses at 29 day gestation survive for anaverage of 44 minutes(13). If anoxia is upheld over thetime of last gasp, permanent brain damage occurs, as shown in dogs, rhesusmonkeys and guinea-pigs(13).  However, oneof the safeguards, to prevent suffering of the foetus, in a report by van derValk et al.

states that the procedure of foetal blood collection must not beginuntil at least 5 minutes after an effective neck cut has been completed andthat the foetus must remain severely anoxic throughout the procedure(14,15). This contradicts a study fromonly two years before by Jochems et al. as they state that discomfort might beexperienced until actual brain damage occurs(4). So, where van der Valk believesthat the foetus will not experience suffering during anoxia, Jochems deems thatsuffering could be experienced during this time until brain damage(4,14). Another safeguard listed in thevan der Valk report states that if the foetus takes a breath then, to avoidsuffering, it must be stunned with a captive bolt. Conversely, in the Jochemset 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 Jochemsclearly states that stunning with a captive bolt would be immoral, yet van derValk 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 Welfareadvisory committee, they support the three safeguards that van der Valk states(15). The guidelines from Mellor canbe seen in Table 2 below.

      Table 2.The Guidelines for the humane slaughter of bovine foetuses as set out by theNational Animal Welfare advisory committee(15). Guideline Explanation 1. Slaughter of the pregnant dam must be humane To ensure the dam remains insensible to pain and distress, the slaughter must meet high welfare standards. 2.

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. 3. Where practical, leave the foetus in the uterus until it is dead All 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 To 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. 6. Foetuses exposed alive may be killed immediately If exposed, the foetus can be killed by a neck cut or by captive bolt destruction of the brain. 7. Unaware (unconscious) foetuses die without suffering Provided the foetus does not inflate their lungs, they cannot become aware and therefore cannot suffer.

     2.3.2    Scientific 2.

3.2.1 Batch-to-batch variation and fraudulentmarketing 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 multipledifferent growth factors or growth inhibition factors could be present. Ultimately,variation in the concentrations of the components can leady to experimentalvariability and as a result limit the inter-laboratory reproducibility of anexperiment(8). In2013, GE Healthcare issues a product information to customers which stated thatbatches of FBS that had been produced by PAA Laboratories between 2008 and 2013could be subject to “label non-conformances”(6). The statement released was: “These products may contain addedadult bovine serum albumin (BSA) of United States origin, water, and/or cellgrowth promoting additives”. This warning from GE Healthcare about the purityof FBS prompts the question of how well the FBS market is regulated.

The USFood and Drug Administration (FDA) reported that 143 batched of FBS wereaffected by this incidence(16). This incidence may have asubstantial impact on thousands of cell culture experiments.Anotherincidence of abuse to the regulations was in 1994 in New Zealand. It wasreported 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 Zealandin that year. Even in 2014, the exact figures for the production rate of FBSthroughout the world was unavailable(6). This raises major suspicions,as there is still a possibility that FBS is being blended with other sera tokeep up with the rising demand from the industry.

 2.3.2.2 ContaminantsAnothermajor disadvantage is that the serum can be contaminated with bacteria,mycoplasmas, fungi, viruses, yeast, endotoxins and immunoglobulins(4). In a study done by Bieback etal. in 2009 found that in four FBS-supplemented cultures, three showedbacterial contamination, meaning it was necessary to discard three of saidcultures(17).   3.  Serum-Free Media 3.

1       Introduction Serum inmedia introduces unknown variables into the cell and tissue culturing, so oneof the main reasons serum-free media (SFM) was introduced is because thecomponents can be defined. SFM can also be cell-specific. There have beenstudies by Barnes and Sato, 1980; Taub, 1990 and Bjare, 1992 investigating variouscombinations of hormones, nutrients and purified proteins to replace serum inspecific cell lines and types(3,18–20). They found that the combinationswere unique to each cell type and therefore in many cases it cannot supplementthe growth of other cell types. The high specificity, that derives from knowingthe composition of the media, gives the opportunity for specific stimulationand differentiation for individual cell types. However, general-purpose serum-free media has not been developed and is likely to be an unachievablegoal.

  3.2       What serum-free media requires Todevelop serum-free media, it is important to understand its function in cellculture. In addition to supplying hormones for growth, serum also providesproteins that bind to vitamins, lipids, metals and hormones(18).

When serum is absent, substitutecomponents must be added to replace the major function in its place. The maincomponents required for a serum-free media are hormones, growth factors,cell-attachment factors and transport binding proteins(14,20). 3.2.1    Serumalbumin Inserum-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 toxiccomponents in the culture medium. Xeno-free formulations typically use humanserum albumin (HSA), however the performance of it can depend on the source asthere are varying methods of isolation from the blood, which in turn may affectthe characteristics of the product.

Therefore, to obtain the optimal productfor a specific cell culture, screening of HSA suppliers is needed. To avoidusing albumin, ACF medium preparations can add fatty acids, lipids,phospholipids and trace elements as a replacement(10,19).  3.

2.2    Insulin Aclinical grade recombinant insulin is most widely used in serum-free formulationsin order to uptake glucose, metabolise lipids and synthesise DNA. It is usuallyin the concentration range of 2-10 mg/L. An alternative to insulin isrecombinant IGF-1 in XF or ACF. Due to the direct activation of the IGF-1receptor, it is used when yielding an enhanced performance with specific celltypes(3,10).  3.

2.3    Humantransferrin Humantransferrin is a carrier protein. Its function is to transport iron into thecell to optimize cell growth and proliferation. Derived from human plasma, itis collected as source plasma and approved for human use.

Recombinantequivalents have limited availability commercially at a higher price withcomparable performance(3). Alternatively, salts such asiron ethylenediaminetetraacetic acid and other iron chelators can be used inACF media. However, there’s a chance that the salts could have negative resultson cell growth, due to free radicals forming and the lack of iron available tothe cells(10,15).  3.2.4    Hormones Glucocorticoids,thyroid hormones and oestrogens can be used in serum-free media and are themost commonly used. An increase in proliferation occurs in adherent cells whenadding hydrocortisone, progesterone and dexamethasone. Specific combinations ofhormones added to ACF media can be a key component, especially when certaincell types are used in cell therapy applications(10,15).

 3.2.5    Growthfactors Growthfactors stimulate cell proliferation and maintain the characteristics of acell.

XF and ACF media use basic fibroblast factor, epidermal growth factor,transforming growth factor beta, vascular endothelial growth factor andplatelet-derived growth factor(3). Available as recombinantproteins, they are widely used for cell therapy. The key to achievingoptimised, cell specific, serum-free ACF medium are specific growth factors,concentrations and synergistic effects. At premium pricing, cGMP growth factorsare manufactured, but are used less often(10,22).  3.

3       Serum-free investigations of specificcell types 3.3.1    Epithelialcells In thepast ten years, epithelial cells have been predominantly cultured in definedmedia 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 mediasupplemented with hormones. The medium also contained epidermal growth factor,hydrocortisone, insulin, transferrin, progesterone, ethanolamine andphosphoethanolamine. The medium was high in calcium, which prompted the cellsto grow less but differentiate more.

Although a mixed inoculum of fibroblasts,keratinocytes and epithelial cells was used, the medium was specificallyselective towards the epithelial cells(21). Another study was conducted byLechner et al. using the same supplements as Tsao, except for progesterone. Thestudy proved that normal human bronchial epithelium cells could be cultivatedin dishes coated with collagen, albumin and fibronectin(22). It hasbeen discovered that epithelial cells grown invitro 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 studygoes on to discover that the cells would respond to hormones and retain theirstructure for several months in the culture, due to the formation of vesicularstructures(23). In contrast, a study byReznikoff et al. demonstrated that epithelial cells grown in Ham’s F12 medium,didn’t improve the growth when compared to media containing serum. This wasproved by the mitogenic reaction when serum was added and an increase indifferentiation of the cells when the concentration of calcium was increased(24).  4.

  Serum Substitutes A studyby Fang et al. investigated the growth capability of five neck and headsquamous carcinoma cells (OECM-1, TW01, HONE-1, SCC25 and FaDu) and onedysplastic oral keratinocyte cell (DOK) lines in six bovine calf serum based cellcultures in comparison to FBS. They were cultured in the FBS alternatives for30 serial passages to determine their ability to support long term growth. Themethods used were assessing the morphology of the cells in each culture, platingefficiency assay and multiple functional assays.  The seraused in addition to FBS were newborn calf serum, bovine calf serum (CS),iron-supplemented calf serum(ICS).

Three bovine serum-based alternatives that wereused fetalgro bovine growth serum(FG), cosmic calf serum(CCS) and foetal clone threeserum(FC3).  Fromtheir results, newborn calf serum containing medium was could not sufficientlysupport the proliferation of the cell lines and therefore were not able tosupport long term growth. In contrast, many vendors actually recommend newborncalf serum as a cheaper replacement for FBS. A previous study discovered thatcertain sera may have reduced cell detachment due to low trypsin inhibitoractivity, but a duplicate study including Accutase (a detachment reagent) stillexhibited poor extension and delayed attachment in newborn calf serum.  In theTW01 cell line, a nasopharyngeal carcinoma cell line, FC3, CCS and FG showedgrowth rates similar to FBS, but CS and ICS had a reduced proliferation andwere more compact than cells grown in FBS. CCS and FG showed slight granularityof the cytosol and the morphology in FC3 appeared similar to that of FBS(25).

 Incontrast, the HONE-1 nasopharyngeal carcinoma cell line cultured in FC3 almostbetter than FBS. CCS and FG showed a reduction in growth in the first tenpassages but in the following tests they grew similarly to FBS. The cellscultured in CS and ICS again showed reduced proliferation but instead showedincreased granularity.

Whereas FG and CCS exhibited as more compacted growth ofHONE-1 cells(25).  The proliferationin the two cell lines TW01 and HONE-1, which are both nasopharyngeal carcinomacell lines, were similar for FC3. However, in TW01 cells, ICS and CS werecompact with CCS and FG showing granularity, but in HONE-1 cells, the cellswere showing granularity for ICS and CS and compact in CCS and FG(25).    5. Conclusion The future of cell culturing requires morein depth investigation into alternatives to foetal bovine serum but of the manytypes of culture supplements, each have their advantages and disadvantages.

This paper has reviewed howfoetal bovine serum works, along with the advantages and disadvantages to usingit for cell culture, and the alternatives available.  Foetalbovine serum does have its advantages as a supplement to cell culture media,however, from this paper, the severity of some of the disadvantages outweighthe advantages. While the worldwide production rate of FBS is unavailable,there will always be an uncertainty to composition of FBS being sold on themarket. If the FBS market was regulated thoroughly, the industry could buy FBSin confidence that it is of high-quality.

Due to the shortage of FBS, several alternatives have been developed. Sera fromother animals, like goats or horses, have been suggested as potentialalternatives, but as a result of them only supporting the growth of a smallamount 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-derivedsupplements is that they are non-xenogeneic when used with human cell lines. However, they are strictly used to culturehuman cell lines only for therapeutic purposes, such as stem cells andmesenchymal stromal cells, due to the limited availability. Panexin is a chemically defined serum replacement forthe cultivation of cells under serum-free culture conditions or tosignificantly reduce the amount of serum used in cell culture. It supports thegrowth of many cell types in an optimum manner without any extra handling whencompared to serum. In addition, as Panexin is fully chemically defined, no lottesting is required.

It contains no growth factors, which means a definedproliferation can be seen(26).As a result, there is a need for research into whetherPanexin is a suitable replacement for foetal bovine serum.  

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