|Foetal Bovine Serum: Background Dr Jan van der Valk 3Rs-Centre Utrecht Life Sciences, Utrecht University||Click here for presentation slides. Dr van der Valk provides information on scientific and animal welfare drawbacks of using FBS.|
|A New Definition for Animal-Free Testing: Replacing Animal-Derived Components in In Vitro Methods Dr Carol Treasure XCellR8||Click here for presentation slides. Dr Treasure describes how to transition cells from media containing FBS to media supplemented with human serum and XCellR8’s experiences in ensuring animal-product-free supply chains in the general context of method development.|
|Foetal Bovine Serum: Replacement Dr Jan van der Valk 3Rs-Centre Utrecht Life Sciences, Utrecht University||Click here for presentation slides. Dr van der Valk examines how the use of alternatives to FBS benefits research, particularly data reproducibility. He discusses the development of chemically defined serum-free media and the use of the FCS-Free Database (fcs-free.org) to find serum-free media for specific cell types.|
|Good In Vitro Method Practices (GIVIMP) and its implementation Dr Sandra Coecke European Commission Joint Research Centre, EURL ECVAM||Click here for presentation slides. Dr Coecke shares insight into the implementation of the OECD Good In Vitro Method Practices (GIVIMP) guidance document.|
- 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
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.
- 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 Scientific Advisory Committee (ESAC) states, “For methods forwarded to ECVAM for validation/prevalidation where [the use of non-animal alternatives to FBS] is not fulfilled, a justification for future use must be provided, including measures taken to seek non-animal alternatives to [FBS].”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.
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.
17ESAC. ESAC statement on the use of FCS and other animal-derived supplements. 2008. https://eurl-ecvam.jrc.ec.europa.eu/about-ecvam/archive-publications/publication/ESAC28_statement_FCS_20080508.pdf.
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.