FBS and other animal components used in cell culture

Animal-derived components in cell culture media, such as serum, hydrolysates, growth factors, hormones, proteins, peptides, lipids, adhesion factors, and amino acids, compromise the quality and reproducibility of research. Particularly, foetal bovine serum (FBS) is composed of an undefined mixture of macromolecules, including hormones, transport proteins, growth factors, lipids, minerals, elements, and detoxifying factors.

As a supplement for cell culture media, FBS is intended to maintain cell viability and facilitate cell metabolism, growth, proliferation, and spreading in culture.^1,2,3 FBS is obtained from the blood of foetal calves. When pregnant cows are slaughtered, a large-gauge needle is used to draw the blood out of the beating heart of the foetus.^1,4 It is estimated that 600,000 litres of FBS – obtained from up to 1.8 million bovine foetuses – are produced worldwide each year,⁵ presenting significant ethical and scientific challenges.

Please see here to download a printable version of a factsheet on alternatives to the use of foetal bovine serum (FBS) in cell culture applications.

Please see here for a 2019 webinar on replacing foetal bovine serum in cell culture media while maintaining robust cell growth and cellular functions.

Scientific and Availability Limitations
Workshops and Presentations on the Replacement of FBS
Application of Alternatives to FBS in Regulatory and Non-regulatory Settings
Media Formulations and Companies that Sell Serum-free Medium or Cell Culture Supplements

SCIENTIFIC AND AVAILABILITY LIMITATIONS

• Variability: Significant batch-to-batch variation exists because serum is a complex mixture of an undefined composition of biomolecules (in which, according to proteomic and metabolomic studies, an estimated 1,800 proteins and more than 4,000 metabolites are present).⁶ The qualitative and quantitative differences in proteins between lots occur as a result of geographical and seasonal variation and may also explain discrepancies among results from in vitro studies that use FBS as the cell culture supplement.^3,7
• Unexpected and undesired outcomes: For instance, it has been shown that serum can suppress TGF-β1, thus preventing chondrogenesis in fibroblast-like type-B synoviocytes.⁸
• Risk of contamination: The use of FBS or other animal-derived substances is especially problematic in the manufacture of biologics for human therapies due to a risk of contamination by animal proteins or pathogens.⁶ In this context, FBS poses a biosafety risk, as it is possible for exogenous agents (i.e. endotoxins, mycoplasma, or viral particles) to contaminate cultured cells or for bovine proteins to contaminate biologics.^1,2,7
• Potential shortage: Due to the extensive use of FBS in vaccine development, research laboratories, and drug production by the expanding biopharmaceutical industry, there is the potential for a future shortage.^3,5

WORKSHOPS AND PRESENTATIONS ON THE REPLACEMENT OF FBS

Because of the scientific and ethical concerns associated with serum collection and use, scientific organisations have held workshops on replacing FBS in in vitro assays.

2003 Workshop Organised by the European Society of Toxicology In Vitro, the Netherlands Organisation for Health Research and Development, and the Swedish National Board for Laboratory Animals This workshop, titled “Towards Better In Vitro Methods: The Replacement of Fetal Bovine Serum” was held to discuss possible ways to reduce the use of FBS in cell culture.⁹ The workshop report recommends that, when possible, FBS be replaced with alternative products, such as chemically defined media supplements. Furthermore, participants recommended making information on alternative supplements more accessible, including through the publication of serum-free media composition by laboratories and companies, regular symposia for discussion of the available alternatives, and the development of resources, such as a public database for cell type–specific serum-free media formulations. They also proposed that registration authorities regard changes to FBS alternatives as minor, making the transition to serum-free media more feasible and encouraging further development and use of chemically defined media.⁹

2009 Workshop Organised by the European Society of Toxicology In Vitro, the Dutch-Belgian Society for In Vitro Methods, and the Danish In Vitro Toxicology Network In 2009, a follow-up workshop was held to discuss ways to further the development and use of serum-free media.³ It stressed the need for laboratories to make the transition to serum-free, chemically defined media and that procedures for the development of such media should be made publicly available. In addition, guidance on developing serum-free media formulations was provided.³

2016 Workshop Organised by the SET Foundation and the German Animal Welfare Federation In this workshop, participants discussed ethical and scientific issues related to FBS, including batch-to-batch variation, interaction with test substances, and unknown composition. In addition, they highlighted issues such as shortcomings of FBS for particular study purposes and significant fraud by FBS suppliers, which has led to a risk of adulterated FBS composition.¹⁰

The workshop report concluded that there is “unmistakable demand for chemically defined media for achieving data reliability and for overcoming the reproducibility crisis caused by FBS charges of non-definable qualities”.¹⁰ It recommended increased funding and continued development of serum-free culture models and the use of serum-free media when establishing new cell lines.

APPLICATION OF ALTERNATIVES TO FBS IN REGULATORY AND NON-REGULATORY SETTINGS

Serum-free media or human platelet lysates can replace FBS when culturing cells. For optimal definition and reproducibility, the goal should be to use chemically-defined animal-free medium, thus avoiding the use of all animal-derived supplements. When culturing cells, researchers should consider the following.

• For some cell types, serum-free media have already been developed, and they are available commercially or their components are published in the literature (see below).^11-14 Guidance is available for adapting cells to serum-free media.^15,16
• For cell lines in which serum-free media have not been developed commercially or published in the literature, researchers will need to optimise the concentration of supplements and transition the cells to the new media. Researchers can test FBS-free media in parallel with their currently used FBS-containing medium to identify an equivalent chemically-defined medium. Thereafter, the concentration of media supplements used should be published in order to facilitate their use in other laboratories.
• When validating tests for regulatory use, animal-component-free medium should be incorporated into any new validation efforts or test guidelines (TGs) using cells cultured in vitro. For example, the EURL ECVAM’s test presubmission form asks applicants to justify why animal derived serum is used and explain if they are considering the possibility to replace it.¹⁷ The Organisation for Economic Co-operation and Development (OECD) has published TGs that use cells maintained in serum-free media, including several involving reconstructed human tissues (e.g. TGs 431, 439, 442D, and 492).^18-21 In fact, numerous three-dimensional reconstructed human tissues, such as those from MatTek, Epithelix, and SkinEthic, are maintained in serum-free medium.

MEDIA FORMULATIONS AND COMPANIES THAT SELL SERUM-FREE MEDIUM OR CELL CULTURE SUPPLEMENTS

For additional FBS-free media formulations and products, see the Fetal Calf Serum-Free Database. Disclaimer: A number of the companies below sell animal-derived products in addition to alternatives to FBS. PETA Science Consortium International e.V. does not endorse any of the organisations listed.

• REFERENCES

  ¹Brunner D, Frank J, Appl H, Schöffl H, Pfaller W, Gstraunthaler G. Serum-free cell culture: The serum-free media interactive online database. ALTEX. 2010;27(1):53-62. https://www.ncbi.nlm.nih.gov/pubmed/20390239.

  ²Gstraunthaler G. Alternatives to the use of fetal bovine serum: Serum-free cell culture. ALTEX. 2003;20(4):275-281. https://www.ncbi.nlm.nih.gov/pubmed/14671707.

  ³Van der Valk J, Brunner D, De Smet K, et al. Optimization of chemically defined cell culture media – replacing fetal bovine serum in mammalian in vitro methods. Toxicol In Vitro. 2010;24(4):1053-1063. https://doi.org/doi:10.1016/j.tiv.2010.03.016.

  ⁴Rauch C, Feifel E, Amann EM, et al. Alternatives to the use of fetal bovine serum: Human platelet lysates as a serum substitute in cell culture media. ALTEX. 2011;28(4):305-316. https://www.ncbi.nlm.nih.gov/pubmed/22130485.

  ⁵Brindley DA, Davie NL, Culme-Seymour EJ, Mason C, Smith DW, Rowley JA. Peak serum: Implications of serum supply for cell therapy manufacturing. Regen Med. 2012;7(1):7-13. https://www.ncbi.nlm.nih.gov/pubmed/22168489.

  ⁶Gstraunthaler G, Lindl T, van der Valk J. A plea to reduce or replace fetal bovine serum in cell culture media. Cytotechnology. 2013;65(5):791-793. https://doi.org/10.1007/s10616-013-9633-8.

  ⁷Van der Valk J, Gstraunthaler G. Fetal Bovine Serum – a pain in the dish? Altern Lab Anim. 2017;45(6):329-332. https://www.researchgate.net/publication/322315373_Fetal_Bovine_Serum_FBS_-_A_pain_in_the_dish.

  ⁸Bilgen B, Orsini E, Aaron RK, Ciombor DM. FBS suppresses TGF- β1-induced chondrogenesis in synoviocyte pellet cultures while dexamethasone and dynamic stimuli are beneficial. J Tissue Eng Regen Med. 2007;1(6):436-442. https://doi.org/10.1002/term.56.

  ⁹Van der Valk J, Mellor D, Brands R, et al. The humane collection of fetal bovine serum and possibilities for serum-free cell and tissue culture. Toxicol In Vitro. 2004;18(1):1-12. https://www.ncbi.nlm.nih.gov/pubmed/14630056.

  ¹⁰Van der Valk J, Bieback K, Buta C, et al. Fetal Bovine Serum (FBS): Past – present – future. ALTEX. 2018;35(1):99-118. https://doi.org/10.14573/altex.1705101.

  ¹¹Chen G, Gulbranson DR, Hou Z, et al. Chemically defined conditions for human iPSC derivation and culture. Nat Methods. 2011;8(5):424-429. https://doi.org/10.1038/nmeth.1593.

  ¹²Burridge PW, Matsa E, Shukla P, et al. Chemically defined generation of human cardiomyocytes. Nat Methods. 2014;11(8):855-860. https://doi.org/10.1038/nmeth.2999.

  ¹³Efe JA, Hilcove S, Kim J, et al. Conversion of mouse fibroblasts into cardiomyocytes using a direct reprogramming strategy. Nat Cell Biol. 2011;13(3):215-222. https://doi.org/10.1038/ncb2164.

  ¹⁴Lu J, Hou R, Booth CJ, Yang SH, Snyder M. Defined culture conditions of human embryonic stem cells. Proc Natl Acad Sci USA. 2006;103(15):5688-5693. https://doi.org/10.1073/pnas.0601383103.

  ¹⁵ThermoFisher Scientific. Adaptation of cell cultures to a serum-free medium. https://www.thermofisher.com/us/en/home/references/protocols/cell-culture/serum-protocol/adaptation-of-cell-cultures-to-a-serum-free-medium.html.

  ¹⁶CELLnTEC. I have early passage cells growing, can I swap them directly into the CELLnTEC medium? http://cellntec.com/products/resources/tech/faq/#weaning.

  ¹⁷European Commission. EURL ECVAM test method submission – test presubmission form. 2022. https://joint-research-centre.ec.europa.eu/eu-reference-laboratory-alternatives-animal-testing-eurl-ecvam/alternative-methods-toxicity-testing/validation-and-submission-process/eurl-ecvam-test-method-submission_en.

  ¹⁸OECD. Test No 431: In Vitro Skin Corrosion: Reconstructed Human Epidermis (Rhe) Test Method. 2015. http://www.oecd-ilibrary.org/environment/test-no-431-in-vitro-skin-corrosion-reconstructed-human-epidermis-rhe-test-method_9789264242753-en.

  ¹⁹OECD. Test No 439: In Vitro Skin Irritation – Reconstructed Human Epidermis Test Method. 2015. http://www.oecd-ilibrary.org/environment/test-no-439-in-vitro-skin-irritation-reconstructed-human-epidermis-test-method_9789264242845-en.

  ²⁰OECD. Test No 442D: In Vitro Skin Sensitisation. 2018. http://www.oecd-ilibrary.org/environment/test-no-442d-in-vitro-skin-sensitisation_9789264229822-en.

  ²¹OECD. Test No 492: Reconstructed Human Cornea-Like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage. 2018. http://www.oecd-ilibrary.org/environment/test-no-492-reconstructed-human-cornea-like-epithelium-rhce-test-method-for-identifying-chemicals-not-requiring-classification-and-labelling-for-eye-irritation-or-serious-eye-damage_9789264242548-en.