Serum-free Media and Serum Alternatives. Jan van der Valk, 3Rs-Centre ULS, DASS, Fac. Vet. Med, UU/ U-AIM

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1 Serum-free Media and Serum Alternatives Jan van der Valk, 3Rs-Centre ULS, DASS, Fac. Vet. Med, UU/ U-AIM

2 Jochems et al., (2002) ATLA 30, Conclusions Jochems et al. Fetuses are likely exposed to pain and/or Policy makers should note that vertebrate fetuses discomfort can and feel therefore pain, current and that practice different of opinions fetal exist blood amongst harvest is scientists inhumane. as to the possible onset of pain experience in vertebrate fetuses. ( )... a relevant guideline may be issued.

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4 Recommendations to avoid foetal suffering Blood collection must not begin until at least 5 min after slaughter Foetus must not be allowed to breathe air after uterus removal Otherwise, foetus must be killed by stunning Foetal Calf Slaughter Welfare Protocol (Mellor & Gregory, 2003) Van der Valk et al (2004) Toxicology In Vitro 18 (1), 1-12

5 In vitro method: alternative? FBS should NOT be used in an in vitro method when that method is developed to replace experimental animals!!

6 Scientific problems Composition of FBS unknown Qualitative and quantitative variations between serum batches May contain different amounts of endotoxins, haemoglobin and other adverse factors May be contaminated with viruses, bacteria, fungi, mycoplasms and prions Reproducibility of experiments Safety of products

7 FBS issues Animal welfare Scientific issues: unknown and variable composition Safety issues Availability/price Fraud

8 Conclusions When considering supplementing cell and tissue culture media with animal serum the Not, unless. principle should be applied. Preferentially, the medium should not contain any animal-derived component, unless it was proved to be an absolute requirement. Van der Valk et al, 2010

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10 OECD it is recommended to develop new in vitro methods with a serum-free, chemically-defined medium, to avoid potential sources of uncertainty that may be introduced by using animal serum (Jochems et al., 2002; Pamies et al., 2016).

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12 Serum free medium Media Serum-free Description Does not require supplementation with serum, but may contain discrete proteins or bulk protein fractions (e.g., animal tissue or plant extracts) and are thus regarded as chemically undefined (see: chemically defined media). Protein-free Does not contain high molecular weight proteins or protein fractions, but may contain peptide fractions (protein hydrolysates), and are thus not chemically defined. Protein-free media facilitate the down-stream processing of recombinant proteins and the isolation of cellular products (e.g., monoclonal antibodies), respectively. Animal-derived component-free Chemically defined Does not contain components of animal or human origin. These media are not necessarily chemically defined (e.g., when they contain bacterial or yeast hydrolysates, or plant extracts). Does not contain proteins, hydrolysates or any other components of unknown composition. Highly purified hormones or growth factors added can be of either animal or plant origin, or are supplemented as recombinant products (see: animal-derived component-free media).

13 Platelet lysates Growth factors Source: safe /clinically tested, high quality Human based xeno-free system Universal application Gstraunthaler et al, 2014

14 Chemically-defined media Medium is selective for specific cell types. Schulz, Workshop Foetal Bovine Serum. Past-Present-Future

15 Commercially available supplements Limited information on ingredients Change of composition without notice Expensive

16 Basal Medium 50:50 (v/v) mixture of DMEM and Ham s nutrient mixture F-12 ITS supplement (insulin, transferrin and selenium) Van der Valk et al, 2010

17 Supplements Hormones Growth factors Protease inhibitors Protein hydrolysates Shear force protectors Proteins Vitamins Amino acids Glutamine Trace elements Lipids Antibiotics Attachment factors Osmolarity Van der Valk et al, 2010

18 Factorial design Applying factorial design approaches have been shown to minimise the screening time, allow prediction for best medium formulation and can be used as a high throughput medium optimisation platform.

19 Adaptation

20 Adaptation

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22 Available information sources Expand commercial with published information Expand with user experiences Free use Funding required

23 FCS-free.org

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26 Actions Raise awareness: scientists funding organisations regulatory authorities National Committees Complete database

27 OECD Batches of serum can differ dramatically in their ability to support the growth of cell lines due to variation in the concentration of growth factors and hormones, therefore, new batches should be tested on the appropriate cell line(s) for cell attachment, spreading, cloning efficiency, growth rates and activity in functional assays (Geraghty et al., 2014). These effects can be overcome by adaption to serum-free culture conditions (Section 4.3.3) using specific protocols (Beltran Paschoal et al., 2014; Leong et al., 2017; Sinacore et al., 2000) for a gradual weaning of cells (van der Valk et al., 2010). it is recommended to develop new in vitro methods with a serum-free, chemically-defined medium, to avoid potential sources of uncertainty that may be introduced by using animal serum (Jochems et al., 2002; Pamies et al., 2016).

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29 Final conclusion Serum free media are: Better for the animals Better for research AWARENESS!!

30 Thank You!

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32 In Vitro Models for Animal Replacement Q & A Session Sponsor