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Fetal bovine serum

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Bottle of FBS for cell culture

Fetal bovine serum (FBS) is the most widely used serum-supplement for the in vitro cell culture of eukaryotic cells. This is due to it having a very low level of antibodies and containing more growth factors, allowing for versatility in many cell culture applications. Fetal bovine serum is derived from the blood drawn from a bovine fetus via a closed system of collection at the slaughterhouse.

The globular protein bovine serum albumin (BSA) is a major component of fetal bovine serum. Besides BSA, fetal bovine serum is a rich source of growth and attachment factors, lipids, hormones, nutrients and electrolytes necessary to support cell growth in culture. It is typically added to basal cell culture medium, such as DMEM or RPMI, at a 5–10% concentration.

Because it is a biological product, FBS is not a fully defined media component, and as such varies in composition between batches.[1] As a result of this and in an attempt to minimize the possibility of transfer of adventitious agents, serum-free and chemically defined media (CDM) have been developed. However, the effectiveness of serum-free media is limited as many cell lines still require serum in order to grow, and many serum-free media formulations can only support the growth of narrowly-defined types of cells.[2]

Production

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FBS is a by-product of the meat industry. FBS, as with the vast majority of animal serum used in cell culture, is produced from blood collected at commercial slaughterhouses from cattle that also supply meat intended for human consumption.[3]

The first stage of the production process for FBS is the harvesting of blood from the bovine fetus after the fetus is removed from the slaughtered cow. The fetus dies from the lack of oxygen by remaining in the protective environment of the uterus for a minimum of 15–20 minutes after the cow is dead.[4] The blood is collected aseptically into a sterile container or blood bag and then allowed to clot. The normal method of collection is cardiac puncture. This minimizes the danger of serum contamination with micro-organisms from the fetus itself, and the environment, while maximizing the volume of blood collected. It is then centrifuged to remove the fibrin clot and the remaining blood cells from the clear yellow (straw) colored serum. The serum is frozen prior to further processing that is necessary to make it suitable for cell culture.[5]

The second stage of processing involves filtration, typically using a filtration chain with the final filtration being three sterile 0.1 micron membrane filters. The aseptically processed FBS is subjected to stringent quality control testing and is supplied with a detailed Certificate of Analysis. The certificate gives full test results and information concerning the origin of the serum. Certificates of Analysis vary between commercial suppliers, but each usually includes the following details: filtration statement, country of blood collection, country of manufacture, cell growth performance testing, microbial sterility testing, as well as screening for mycoplasma and virus, endotoxin, hemoglobin, IgG gamma glutamyl transferase[6] and total protein assays. FBS may also be tested for country of collection.[7]

Ethics

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Ethical questions have been raised regarding the blood collection process due to the potential suffering caused to the fetus. There has been discussion concerning the methodologies for the collection of fetal bovine serum. The International Serum Industry Association (ISIA) has published literature[8][9] providing detail on the extensive regulation and processes employed to ensure that serum is collected in an ethical manner. Although the act of slaughter of the dam and the time which passes in the slaughter process prior to harvesting will induce unconsciousness or death of the fetus prior to serum harvesting, it has been postulated that exposure of live unborn calves to oxygen could cause them to gain awareness before being killed, resulting in active debate about the ethics of harvesting serum.[10][11] While the Industry Association accepts that certain organizations have concerns, they maintain that all collections of serum take place under veterinary supervision in registered slaughterhouses controlled by the competent authority in the country of collection.

Serum use

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Fetal bovine serum
Frozen fetal bovine serum

Fetal bovine serum is commercially available from many manufacturers, and because cells grown in vitro are highly sensitive, customers usually test specific batches to check for suitability for their specific cell type. When changing from batch to batch it is usual to adapt the cells to the new batch of material, for example, by mixing 50% of the old serum with 50% of the new serum and allowing the cells to acclimatize to the new material.

Serum is stored frozen to preserve the stability of components such as growth factors. When serum is thawed, some precipitation may be seen. This is a normal phenomenon, and it does not compromise the quality of serum in any way. The precipitate may be removed by transferring the serum to sterile tubes and centrifuging for 5 minutes at 400 × g. To limit the amount of precipitation, it is recommended that the serum is thawed in a refrigerator at 2-8˚C. The serum should be regularly stirred during this process. Repeated freeze/thaw cycles should be avoided, and it is advisable to dispense the serum into single use aliquots before freezing.[12]

Source history

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Serum produced for use in the biotechnology industry and research sectors is highly regulated. The collection and movement of all animal derived products globally is strictly controlled. Veterinary control of animal derived products largely follows the regulations set by the EU (DG SANCO) and the US (USDA). The current regulation governing the importation of animal by-products into the EU is covered by Regulation (EU) 1069/2009 and the implementation document Regulation (EU) 142/2011.

FBS is a product collected worldwide with the main collections being centered in the United States, Australia, New Zealand, Canada, Central America, South America, and Europe.

Global sales

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Sales of FBS in 2008 were estimated to be 700,000 liters globally.[citation needed] A 2012 source estimates that about 600,000 liters are made annually, one third of which is suitable for pharmaceutical use under Good Manufacturing Practice. 1 to 3 fetuses are required to produce one liter of serum.[13]

Replacements

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Proposed replacements for FBS include:

  • Bovine ocular fluid, also a byproduct of slaughterhouses. Also contains a variety of mammalian growth factors.[14]
  • Hydrolyzed sericin, a normally discarded byproduct of silk production. Also supports the growth of skin cells.[14]
  • Human platelet lysate (HPL), which can be produced from old stock from blood banks. Contains a variety of mammalian growth factors. Human adipose tissue grows better in HPL than in FBS.[14]
  • Earthworm heat inactivated coelomic fluid, which acts like "blood" in earthworms. Rich in nutrients and growth hormones. Does not contain fibronectin, so an attachment factor must be added.[14]

See also

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References

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  1. ^ Dirks WG (2021). "Ethical Challenges Using Human Tumor Cell Lines in Cancer Research". Ethical Challenges in Cancer Diagnosis and Therapy. Recent Results in Cancer Research. Vol. 218. pp. 39–46. doi:10.1007/978-3-030-63749-1_4. ISBN 978-3-030-63748-4. PMID 34019161. S2CID 235072805.
  2. ^ Yao T, Asayama Y (April 2017). "Animal-cell culture media: History, characteristics, and current issues". Reproductive Medicine and Biology. 16 (2): 99–117. doi:10.1002/rmb2.12024. PMC 5661806. PMID 29259457.
  3. ^ "Terrestrial Animal Health Code". Retrieved 2021-12-10.
  4. ^ Hawkes, Percy (January 2019). "Fetal Bovine Serum and the Slaughter of Pregnant Cows: Animal Welfare and Ethics". Bioprocessing Journal. 18: 4. doi:10.12665/J18OA.Hawkes. Retrieved 8 January 2024.
  5. ^ "Characterization and Qualification of Cell Substrates and Other Biological Materials Used in the Production of Viral Vaccines for Infectious Disease Indications". Food and Drug Administration. Retrieved 2021-12-10.
  6. ^ Cheever M, Master A, Versteegen R (22 December 2017). "A Method for Differentiating Fetal Bovine Serum from Newborn Calf Serum". BioProcessing Journal. 16. doi:10.12665/J16OA.CHEEVER.
  7. ^ Versteegen R, Shatova O, Lind S, Linterman K (15 May 2019). "Testing for Geographic Origin of Fetal Bovine Serum". BioProcessing Journal. 18. doi:10.12665/J18OA.Versteegen. S2CID 182766915.
  8. ^ Versteegen RJ, Murray J, Doelger S (2021). "Animal welfare and ethics in the collection of fetal blood for the production of fetal bovine serum". Altex. 38 (2): 319–323. doi:10.14573/altex.2101271. PMID 33871036. S2CID 233298304.
  9. ^ van der Valk J, Brunner D, De Smet K, Fex Svenningsen A, Honegger P, Knudsen LE, et al. (June 2010). "Optimization of chemically defined cell culture media--replacing fetal bovine serum in mammalian in vitro methods". Toxicology in Vitro. 24 (4): 1053–1063. doi:10.1016/j.tiv.2010.03.016. hdl:1874/191398. PMID 20362047. S2CID 205410680.
  10. ^ Mellor DJ, Gregory NG (February 2003). "Responsiveness, behavioural arousal and awareness in fetal and newborn lambs: experimental, practical and therapeutic implications". New Zealand Veterinary Journal. 51 (1): 2–13. doi:10.1080/00480169.2003.36323. PMID 16032283. S2CID 32259690.
  11. ^ Jochems CE, van der Valk JB, Stafleu FR, Baumans V (March 2002). "The use of fetal bovine serum: ethical or scientific problem?". Alternatives to Laboratory Animals. 30 (2): 219–227. doi:10.1177/026119290203000208. PMID 11971757. S2CID 27361021.
  12. ^ Hillebrand JJ, Heijboer AC, Endert E (February 2003). "Effects of repeated freeze-thaw cycles on endocrine parameters in plasma and serum". Annals of Clinical Biochemistry. 54 (2): 289–292. doi:10.1080/00480169.2003.36323. PMID 16032283. S2CID 32259690.
  13. ^ Brindley, DA; Davie, NL; Culme-Seymour, EJ; Mason, C; Smith, DW; Rowley, JA (January 2012). "Peak serum: implications of serum supply for cell therapy manufacturing". Regenerative Medicine. 7 (1): 7–13. doi:10.2217/rme.11.112. PMID 22168489.
  14. ^ a b c d Subbiahanadar Chelladurai, K; Selvan Christyraj, JD; Rajagopalan, K; Yesudhason, BV; Venkatachalam, S; Mohan, M; Chellathurai Vasantha, N; Selvan Christyraj, JRS (August 2021). "Alternative to FBS in animal cell culture - An overview and future perspective". Heliyon. 7 (8): e07686. doi:10.1016/j.heliyon.2021.e07686. PMC 8349753. PMID 34401573.
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