1. IntroductionCell 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.2. Foetal Bovine Serum 2.1 The composition of FBSFBS 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 FBSFBS 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 FBS2.3.1 EthicalDuring 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.2.3.2 Scientific184.108.40.206 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. 220.127.116.11 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 Media3.1 IntroductionSerum 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 requiresTo 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 albuminIn 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 InsulinA 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 transferrinHuman 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 HormonesGlucocorticoids, 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 factorsGrowth 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 types3.3.1 Epithelial cellsIn 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 SubstitutesA 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. 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. 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.5. ConclusionThe 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(25).As a result, there is a need for research into whether Panexin is a suitable replacement for foetal bovine serum.