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Fetal bovine serum (FBS): Past – present – future

August 2017
ALTEX 35(1)

DOI:10.14573/altex.1705101

Authors:

J.B.F. Van der Valk

Utrecht University

Karen Bieback

Heidelberg University

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The supplementation of culture medium with fetal bovine serum (FBS, also referred to as 'fetal calf serum') is still common practice in cell culture applications. Due to a number of disadvantages in terms of quality and reproducibility of in vitro data, animal welfare concerns, and in light of recent cases of fraudulent marketing, the search for alternatives and the development of serum-free medium formulations gained global attention. Here, we report on the 3rd Workshop on FBS, Serum Alternatives and Serum-free Media, where (a) regulatory aspects, (b) the serum dilemma, (c) alternatives to FBS, (d) case-studies of serum-free in vitro applications, and (e) the establishment of serum-free databases, were discussed. The whole process of obtaining blood from a living calf fetus to using the FBS produced from it for scientific purposes is de facto not yet legally regulated, despite the existing EU-Directive 2010/63/EU on the use of animals for scientific purposes. Together with above mentioned challenges, several strategies have been developed to reduce or replace FBS in cell culture media in terms of the 3Rs (Refinement, Reduction, Replacement). Most recently, releasates of activated human donor thrombocytes (human platelet lysates) have been shown to be one of the most promising serum alternatives when chemically defined media are not yet an option. Additionally, new developments in cell-based assay techniques, advanced organ-on-chip and microphysiological systems are covered in this report. Chemically-defined serum-free media are shown to be the ultimate goal for the majority of culture systems, and examples are discussed.

Scanning electron micrograph of resting (left) and activated (right) human thrombocytes, immobilized on sterile filters When platelets are activated, they undergo characteristic morphological changes and form long pseudopodia to adhere to the sites of vessel wall injury. In addition, thrombocytic granule factors are released by exocytosis: vasoactive peptides, factors for initiating and maintaining the clotting cascade, and growth factors for subsequent wound healing. Filters from Millipore Inc., pore size 0.22 µm. Micrograph taken by G. Gstraunthaler.

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Test for the eye irritation potential of seven dilutions of sodium dodecyl sulphate toward L929 fibroblasts A: extracellular acidification rate (EAR), B: modulus of impedance (ІZІ)). The first five recordings of the basal rates are shown, followed by seven dilutions of sodium dodecyl sulphate.

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Test for the eye irritation potential of seven dilutions of sodium dodecyl sulfate toward L929 fibroblasts Left: extracellular acidification rate (EAR), right: modulus of impedance (IZI). The first five recordings of the basal rates are shown, followed by seven dilutions of sodium dodecyl sulfate.

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Schematic view of a 4-organ microfluidic platform (a) Schematic view of the microfluidic platform showing the different cell compartments of a 4-organ system. The system contained two holders for the separate culture devices. Total fluid volume was approximately 4 mL between the chambers and reservoirs. The sizes of the culture compartments were 35.8 x 18.4 x 0.3 mm for Chambers 1, 2, 3 and 29.8 x 15.4 x 0.7 mm for Chambers 4 and 5. The connecting channel dimensions were 5.7 x 1 x 0.3 mm. (b) Shear stress distribution in each compartment of the system. Reproduced with permission from (Oleaga et al., 2016).

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ALTEX 35 99 2018

Data

January 2018

J.B.F. Van der Valk · Karen Bieback · Christiane Buta · Brett Cochrane · Gerhard Gstraunthaler

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Fetal Bovine Serum (FBS): Past - Present - Future; ALTEX 35(1): 99-118 (2018)

Data

January 2018

J.B.F. Van der Valk · Karen Bieback · Christiane Buta · Brett Cochrane · Gerhard Gstraunthaler

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Animal origins free products in cell culture media: a new frontier

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Dec 2024
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Despite the importance of finding replacements for fetal bovine serum (FBS), very few studies have focused on this subject. Historically, the use of animals and their derivatives in growth, reproduction, and physiological studies has raised several concerns. The supplementation of culture media with FBS, also known as fetal calf serum, continues to be widespread, despite its limitations in quality, reproducibility, and implications for animal welfare. Moreover, the presence of counterfeit and illegal products can adversely affect cell cultures and treatments, prompting the search for alternative solutions. To reduce reliance on FBS, various substitutes have been introduced, such as plant-derived proteins, bovine eye fluid, sericin protein, human platelet lysate, and inactivated coelomic fluid, which can provide roles similar to that of FBS. Therefore, it is essential to develop serum-free and animal supplement-free environments suitable for therapeutic and clinical applications, tailored to the specific needs of different cell types. Among the alternatives, plant-based options have gained attention as sustainable and ethical solutions. These include plant-derived peptones from sources like soy and wheat, which are rich in amino acids and peptides essential for mammalian cell growth, as well as plant protein hydrolysates from beans and peas that serve as sources of amino acids and growth factors. Plant extracts, especially from soy and various seeds, contain necessary proteins and growth factors, while phytohormones such as cytokinins and plant polysaccharides can help regulate cell growth. While these alternatives offer benefits like reduced costs and lower risks of disease transmission, further research is necessary to refine and align them with the specific requirements of diverse cell types. Graphical abstract

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The demand for skin models as alternatives to animal testing has grown due to ethical concerns and the need for accurate substance evaluation. These alternatives, known as New Approach Methodologies (NAMs), are increasingly used for regulatory decisions. Current skin models from primary human cells often rely on bovine collagen, raising ethical issues. This study explores self-assembled skin models (SASM) as a new method, utilizing hair follicle-derived keratinocytes reprogrammed into induced pluripotent stem cells (iPSC) and differentiated into fibroblasts and keratinocytes. The model relies on the ability of fibroblasts to secrete collagen to produce a xeno-free dermal layer and on the differentiation of keratinocytes to create a functional epidermal layer. These layers exhibited confirmed metabolic activity and the capability to withstand test substances. The successful development of SASM underscores the significance of accurate alternatives in dermatological research, providing an ethical and reliable option for substance evaluation and regulatory testing.

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Standard information reporting helps to ensure that assay conditions and data are consistently reported and to facilitate inter-laboratory comparisons. Here, we present recommendations on minimum information for reporting on the TEER (trans-epithelial/endothelial electrical resistance) assay (MIRTA). The TEER assay is extensively used to evaluate the health of an epithelial/endothelial cell culture model and as an indicator of the potential toxicity of a test substance. This publication is the result of an international collaboration─called the RespTox (Respiratory Toxicity) Collaborative─through which twelve laboratories shared their protocols for assessing the barrier function of respiratory epithelial cells using the TEER assay following exposure to substances. The protocols from each laboratory were reviewed to identify general steps for performing the TEER assay, interlaboratory differences between steps, the rationale for differences, whether these differences impact results or cross-laboratory comparisons between TEER measurements. While the MIRTA recommendations are focused on respiratory epithelial cell systems, these recommendations can be adapted for other cell systems that form barriers. The use of these recommendations will support data transparency and reproducibility, reduce challenges in data interpretation, enable cross-laboratory comparisons, help assess study quality, and facilitate the incorporation of the TEER assay into national and international testing guidance.

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Fetal Bovine Serum (FBS) is an important ingredient in cell culture media and the current standard for most cells in vitro. However, the use of FBS is controversial for several reasons, including ethical concerns, political, and societal pressure, as well as scientific problems due to the undefined and variable nature of FBS. Nevertheless, scientists hesitate to change the paradigm without solid data de-risking the switch of their assays to alternatives. In this study, HepG2 cells, a human hepatoblastoma cell line commonly used to study drug hepatotoxicity, were adapted to serum-free conditions by using different commercially available media and FBS replacements. After transition to these new culture conditions, the success of adaptation was determined based on cell morphology and growth characteristics. Long-term culturing capacity for each medium was defined as the number of passages HepG2 cells could be cultured without any alterations in morphology or growth behavior. Two media (Advanced DMEM/F12 from ThermoFisher and TCM® Serum Replacement from MP Biomedicals) showed a long-term cultivation capacity comparable to media containing FBS and were selected for further analysis. Both media can be characterized as serum-free, however still contain animal-derived components: bovine serum albumin (both media) and bovine transferrin (only TCM® serum replacement). To assess the functionality of the cells cultivated in either of the two media, HepG2 cells were treated with reference compounds, specifically selected for their known hepatotoxicity characteristics in man. Different toxicological assays focusing on viability, mitochondrial toxicity, oxidative stress, and intracellular drug response were performed. Throughout the different assays, response to reference compounds was comparable, with a slightly higher sensitivity of serum-free cultivated HepG2 cells when assessing viability/cell death and a lower sensitivity towards oxidative stress. Taken together, the two selected media were shown to support growth, morphology, and function of serum-free cultivated HepG2 cells in the early preclinical safety space. Therefore, these results can serve as a starting point to further optimize culture conditions with the goal to remove any remaining animal-derived components.

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Introduction As part of a wider programme of work developing next-generation risk assessment approaches (NGRA) using non-animal methods (NAMs) for safety assessment of materials, Unilever SEAC is exploring the use of a peripheral blood mononuclear cell (PBMC) system to investigate how cells from different arms of the human immune system are impacted by different treatments. To maximise human relevance, the cell cultures are supported by human serum, but this came with some challenges, including an inability to measure induced levels of immunoglobulins due to high background levels. Therefore, a study comparing use of human sera containing media with three different chemically defined serum-free media was undertaken. Materials and Methods PBMC were isolated from healthy donors and cultured in the absence (media alone) or presence of stimulation reagents (CpG-ODN plus IL-15, Pokeweed Mitogen (PWM) or Cytostim (CS)), in RPMI plus human serum, AIM-V, CTS OpTmizer T cell expansion SFM or X-VIVO 15 media. T cell (CD4⁺ and CD8⁺) and B cell proliferation and viability were measured after 6 days, along with levels of total IgG in the cell culture supernatants. Results Each of the serum-free media tested supported good levels of viable and proliferating T cells and B cells over the 6 days of culture, with only a few, small differences across the media, when there was no stimulation. They also enabled detection of a stimulation-evoked increase in IgG levels. There were however some differences in the viability and proliferation responses of T and B cells, to different stimuli, across the different media. Discussion The serum-free media formulations tested in this study offer defined systems for. measuring B cell IgG responses, in vitro, in either a ‘T cell-independent’ (CpG + IL-15) or “T cell-dependent” (PWM or CS) manner and for assessing B cell proliferation, particularly in response to a “T cell-independent” stimulus. However, there are some characteristics and features endowed by human serum that appear to be missing. Therefore, further work is required to optimise animal-free, chemically defined culture conditions for PBMC based assays for inclusion in tiered safety assessments.

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Fetal bovine serum (FBS) accounts for the largest portion of the cost of cultured meat production or cell culture experiments and is highly controversial in terms of animal welfare because it is taken from the fetus of a pregnant cow during slaughtering. Nevertheless, FBS is the most important supplement in the cell culture manufacturing process. This study aimed to develop an FBS substitute from slaughterhouse waste blood to reduce the cost of FBS in cultured meat production through various experiments. Our study successfully demonstrated that adult livestock blood obtained from slaughterhouses can effectively replace FBS. Our substitute, when cultured with bovine myosatellite cells, demonstrated cell growth that was either equivalent to or superior to that of commercial FBS. In the process of muscle generation through differentiation, the substitutes from bovine and chicken formed 70%–75% more bovine muscle compared to the control group using FBS. Furthermore, using the FBS substitute can reduce cell culture costs by approximately 61% compared to using commercial FBS. Therefore, the groundbreaking FBS substitute will not only contribute to the development of technology to mass‐produce cultured meat using livestock byproducts but will also lower the production cost of media for experimental cell culture or vaccine production.

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In the past decades, significant advancement has been made in plant-based platforms for vaccine and pharmaceutical production. Various plant species, including tobacco, corn, potato, and others, have been engineered to produce a wide range of bioactives such as vaccines. Nowadays, with global changes challenging food security for humans, attention of researchers has moved to the use of plant as biotechnological systems for production of nutritious proteins for human nutrition, including animal proteins, that have higher biological values than those from other sources. Although plant-based platforms have considerable advantages over traditional systems such as bacterial and animal systems, there are several obstacles to commercial-scale production, first of all the purification process of the proteins from the expression systems and their structural characterization to ensure their safety and regulatory approval. In this chapter, the issues connected to the production of proteins in plant seeds and a pipeline for their isolation and analysis for proteomics characterization are detailed.

Chemisch definiert – ein zellbasierter Zytotoxizitätsassay ohne fötales Kälberserum

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Tests for cytotoxicity–respectively biocompatibility–are performed e. g. for biological evaluation of medical devices or during the development of microphysiometric systems. Here, often cell culture methods which involve the use of fetal bovine serum (FBS) are employed. However, using FBS raises scientific and ethical concerns. In the presented work a protocol for a chemically defined test for cytotoxicity is described.

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p>Numerous variables can torpedo attempts to replicate cell experiments, from the batch of serum to the shape of growth plates. But there are ways to ensure reliability.</p