ArticlePDF Available

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

Authors:

Abstract and Figures

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.
Content may be subject to copyright.
A preview of the PDF is not available
... FCS can contain substances such as growth factors, hormones, vitamins, and transport proteins, which can potentially influence cell proliferation and maintenance. The use of DMEM without FCS helps to reduce this influence on the experimental setup [12,13]. The cells were further incubated for 12 h. ...
Article
Full-text available
Purpose In the following work, we investigated the effect of matcha green tea extract (MTE) on MCF-7 breast cancer cell viability and estrogen receptor-beta expression (ERβ). Methods MCF-7 cells were stimulated with MTE at concentrations of 5 and 10 µg/ml. Cell viability was assessed using a water-soluble tetrazolium assay (WST-1 assay) after an incubation time of 72 h. ERβ was quantified at gene level by real-time polymerase chain reaction (PCR). A western blot (WB) was carried out for the qualitative assessment of the expression behavior of on a protein level. Results The WST-1 test showed a significant inhibition of viability in MFC-7 cells after 72 h at 10 µg/ml. The WB demonstrated a significant quantitative decrease of ERβ at protein level with MTE concentrations of 10 µg/ml. In contrast, the PCR did not result in significant downregulation of ERβ. Conclusion MTE decreases the cell viability of MCF-7 cells and furthermore leads to a decrease of ERβ at protein level.
... Due to the large number of signaling molecules and metabolic peculiarities for in vitro cultures of embryos, media with more complex additives are applied that can be used via co-cultures (Van der Weijden et al., 2017;Carvalho et al., 2017) or obtained directly from the animal (such as follicular fluid, oviduct fluid, serum) ( Van der Valk et al., 2018;). Other, more sophisticated, systems mimic the microtubular anatomy of the Fallopian tube (Beebe et al., 2002). ...
... Dulbecco's Modified Eagle Medium (DMEM): for supporting the growth of mammalian cells [29,30]; Trypan blue stain: to estimate the number of dead cells in a viable population; 0.25% Trypsin-EDTA solution: to detach adherent cell from culture vessel surfaces [31]; Fetal Bovine Serum (FBS): growth supplement used for cell culture media [32]; Antibiotic-antimycotic (Ab/Am) solution (a mixture of penicillin, streptomycin [33][34][35], and Gibco Amphotericin B to prevent contamination) [36]; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide tetrazolium salt (MTT): for measuring cell proliferation; PBS (Phosphate Buffered Saline): to maintain a constant pH [37,38]. ...
Article
Full-text available
Several innovative materials' properties are predicted using in silicon investigations, a computer simulation technique. 1,2,4,5-tetraazaspiro[5.5]undecane-3-thione was synthesized and characterized by using a variety of spectroscopic approaches followed by in silicon studies. DFT at the B3LYP functional level and the basis set 6-311 ++G (d,p) was used. The title molecule was investigated for its electric dipole moment, polarizability, and static hyperpolarizability. In silicon studies of the compound were done and showed good result to predict pharmacokinetics, lipophilicity, physicochemical and medicinal properties of the target molecule. Molecular docking was done between receptors and ligand by simulating molecular interactions in BRD4 proteins. The compound was also bio-evaluated for its anti-lung cancer activity against A549 cancer cell line. Theoretical calculation of ADMET properties suggested that the synthesized compound shows good pharmacokinetic profile. The results of cytotoxic evaluation indicated that the compound has broad-spectrum cytotoxic activity with IC50 values of 300 μM against A549 cell line.
... These adjustments could potentially restrict the use of certain cell types as not all serum-free media formulations are suitable for all cell origins. Consequently, this limitation may hinder large-scale EV production and increase costs [84,85]. ...
Article
Full-text available
Extracellular vesicle (EV) research has expanded substantially over the years. EVs have been identified in all living organisms and are produced and released as a means of intercellular communication or as a defense mechanism. Recently, nano-scaled vesicles were successfully isolated from edible plant sources. Plant-derived EVs, referred to here as phytosomes, are of a size reported to range between 30 nm and 120 nm in diameter, similar to small mammalian extracellular vesicles, and carry various bioactive molecules such as mRNA, proteins, miRNA and lipids. Due to the availability of many plants, phytosomes can be easily isolated on a large scale. The methods developed for EV isolation from mammalian cells have been successfully applied for isolation and purification of phytosomes. The therapeutic effects of phytosomes on different disease models, such as inflammation and autoimmune disease, have been reported, and a handful of studies have suggested their therapeutic effects on cancer diseases. Overall, the research on phytosomes is still in its infancy and requires more exploration. This review will narrate the anti-cancer activity and characteristics of phytosomes derived from edible plants as well as describe studies which have utilized phytosomes as drug delivery vehicles for cancer with the ultimate objective of significantly reducing the adverse effects associated with conventional therapeutic approaches.
... In order to contribute to the 3R (reduce, refine, replace) principles, increasing attention goes to the development of animal-free chemicallydefined alternatives for FCS. Several synthetic supplements have been tested but did not allow various cell types to proliferate and differentiate properly (van der Valk et al., 2018). Human platelet lysate seems a promising substitute for FCS. ...
Article
Bile acid homeostasis is vital for numerous metabolic and immune functions in humans. The enterohepatic circulation of bile acids is extremely efficient, with ~95% of the intestinal bile acids being reabsorbed. Disturbing intestinal bile acid uptake is expected to substantially affect intestinal and systemic bile acid levels. Here, we aimed to predict the effects of apical sodium-dependent bile acid transporter (ASBT)-inhibition on systemic plasma levels. For this, we combined the in vitro Caco-2 cell transport assays with physiologically based (PBK) modeling. For this proof-of-principle study we used the selective ASBT-inhibitor odevixibat (ODE) as a model compound. Caco-2 cells grown on culture inserts were used to obtain transport kinetic parameters of glycocholic acid (GCA). The apparent Michaelis Menten constant (Km,app), apparent maximal intestinal transport rate (Vmax,app) and ODE's inhibitory constant (Ki) were determined for GCA. These kinetic parameters were incorporated in a PBK model and used to predict the ASBT inhibition effects on plasma bile acid levels. GCA is transported over Caco-2 cells in an active and sodium-dependent manner, indicating the presence of functional ASBT. ODE inhibited GCA transport dose-dependently. The PBK model predicted that oral doses of ODE reduced conjugated bile acid levels in plasma. Our simulations match in vivo data and provide a first proof-of-principle for the incorporation of active intestinal bile acid uptake in a bile acid PBK model. This approach could in future be of use to predict the effects of other ASBT-inhibitors on plasma and intestinal bile acid levels.
... FCS can contain substances such as growth factors, hormones, vitamins, and transport proteins, which can potentially in uence cell proliferation and maintenance. The use of DMEM without FCS helps to reduce this in uence on the experimental setup [12,13]. The cells were further incubated for 12 hours. ...
Preprint
Full-text available
Purpose: In the following work, we investigated the effect of matcha green tea extract (MTE) on MCF-7 breast cancer cell viability and estrogen receptor beta expression (ERβ). Methods: MCF-7 cells were stimulated with MTE at concentrations of 5 and 10 µg/ml. Cell viability was assessed using a WST-1 assay after an incubation time of 72 h. ERβ was quantified at gene level by real-time polymerase chain reaction (PCR). A western blot (WB) was carried out for the qualitative assessment of the expression behavior of on a protein level. Results: The WST-1 test showed a significant inhibition of viability in MFC-7 cells after 72 h at 10 µg/ml. The WB demonstrated a significant quantitative decrease of ERβ at protein level with MTE concentrations of 10 µg/ml. In contrast the PCR did not result in significant downregulation of ERβ. Conclusion: MTE decreases the cell viability of MCF-7 cells and furthermore leads to a decrease of ERβ at protein level.
Preprint
Full-text available
The ability of newly developed drugs to navigate the current translational pipeline is extremely poor, with less than 10% of drugs making this transition even after entry into clinical trials. There are many reasons for this, but interspecies differences in functional and physiological parameters contribute to the problem. Improving the humanrelevance of early pre-clinical in vitro models may help translatability, especially when targeting more nuanced species-specific cell processes. We aimed to define a set of guidelines for effective transition of human primary cells of multiple lineages into a more physiologically relevant, translatable, animal-free culture environment by systematic replacement of animal-derived biomaterials in in vitro culture systems, followed by assessment of effects on cell kinetics and phenotype. We successfully eliminated animalderived biomaterial from primary human dermal fibroblast, uterine fibroblast, pulmonary fibroblast, retinal endothelial cell, and peripheral blood mononuclear cell culture systems and defined the individual requirements of each cell subtype for transition to animal-component free culture conditions. We therefore demonstrate that it is possible to transition (“humanise”) a diverse set of human primary cell types by following a set of simple overarching principals that inform the selection, and guide the evaluation of new, improved, human-relevant culture conditions.
Article
Full-text available
Background: Fetal bovine serum (FBS) is the most used supplement in culture media; however, it may interfere with in vitro assays via effects on cell proliferation and cytokine production. The ideal FBS concentration for assays using apical papilla cells (APCs) remains unknown. Therefore, this study aimed to evaluate the effects of FBS on APC activation, cell viability/proliferation, and cytokine production. Methodology: Human APCs were cultured, plated, and maintained in media containing increasing concentrations of FBS for 24 h, 48 h, 72 h, 7 days, and 14 days in the presence of Lipopolysaccharide (LPS - 1 µg/mL). At each time point, the cells were subjected to the MTT assay. The cytokines transforming growth factor (TGF)-β1, osteoprotegerin (OPG), and interleukin (IL)-6, along with the chemokine CCL2, were quantified using the enzyme-linked immunosorbent assay at the 24-h time-point. Statistical analysis was performed using two-way analysis of variance (ANOVA) followed by Tukey's post-hoc test (p<0.05). Results: In general, APCs exhibited increasing metabolic activity in an FBS concentration-dependent fashion, regardless of the presence of LPS. In contrast, FBS interfered with the production of all the cytokines evaluated in this study, affecting the response induced by the presence of LPS. Conclusion: FBS increased APC metabolism in a concentration-dependent manner and differentially affected the production of TGF-β1, OPG, IL-6, and CCL2 by APCs in vitro.
Article
Full-text available
Introduction: In vitro approaches are an essential tool in screening for toxicity of new chemicals, products and therapeutics. To increase the reproducibility and human relevance of these in vitro assessments, it is advocated to remove animal-derived products such as foetal bovine serum (FBS) from the cell culture system. Currently, FBS is routinely used as a supplement in cell culture medium, but batch-to-batch variability may introduce inconsistency in inter- and intra-lab assessments. Several chemically defined serum replacements (CDSR) have been developed to provide an alternative to FBS, but not every cell line adapts easily and successfully to CDSR-supplemented medium, and the long-term effect on cell characteristics remains uncertain. Aim: The aim of this study was to adapt the TK6 cell line to animal-product free CDSR-supplemented medium and evaluate the long-term effects on cell health, growth, morphology, phenotype, and function. This included a provisional assessment to determine the suitability of the transitioned cell line for standardised genotoxicity testing using the “ in vitro mammalian cell micronucleus test” (OECD TG 487). Materials and methods: Gradual adaptation and direct adaptation methodologies were compared by assessing the cell proliferation, size and viability every passage until the cells were fully adapted to animal-free CDSR. The metabolic activity and membrane integrity was assessed every 4-8 passages by PrestoBlue and CytoTox-ONE™ Homogeneous Membrane Integrity Assay respectively. A detailed morphology study by high content imaging was performed and the expression of cell surface markers (CD19 and CD20) was conducted via flow cytometry to assess the potential for phenotypic drift during longer term culture of TK6 in animal-free conditions. Finally, functionality of cells in the OECD TG 487 assay was evaluated. Results: The baseline characteristics of TK6 cells cultured in FBS-supplemented medium were established and variability among passages was used to set up acceptance criteria for CDSR adapted cells. TK6 were adapted to CDSR supplemented medium either via direct or gradual transition reducing from 10% v/v FBS to 0% v/v FBS. The cell growth rate was compromised in the direct adaptation and therefore the gradual adaptation was preferred to investigate the long-term effects of animal-free CDSR on TK6 cells. The new animal cells showed comparable ( p > 0.05) viability and cell size as the parent FBS-supplemented cells, with the exception of growth rate. The new animal free cells showed a lag phase double the length of the original cells. Cell morphology (cellular and nuclear area, sphericity) and phenotype (CD19 and CD20 surface markers) were in line ( p > 0.05) with the original cells. The new cells cultured in CDSR-supplemented medium performed satisfactory in a pilot OECD TG 487 assay with compounds not requiring metabolic activation. Conclusion: TK6 cells were successfully transitioned to FBS- and animal product-free medium. The new cell cultures were viable and mimicked the characteristics of FBS-cultured cells. The gradual transition methodology utilised in this study can also be applied to other cell lines of interest. Maintaining cells in CDSR-supplemented medium eliminates variability from FBS, which in turn is likely to increase the reproducibility of in vitro experiments. Furthermore, removal of animal derived products from cell culture techniques is likely to increase the human relevance of in vitro methodologies.
Article
Full-text available
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.
Article
PURPOSE: This review of milestones in eye banking describes efforts by the eye banking community to ensure the quality and quantity of corneal tissue over the last century and anticipates key challenges going forward. METHODS: This account draws on a review of the scientific literature, public documents, and eye banking statistics and is augmented by the recollection of the author, discussions with eye bankers, and an analysis of pressing medical and nonmedical issues that will bear on the future success of eye banking in the United States and internationally. RESULTS: The author identifies five eras of eye banking, highlighting scientific breakthroughs in surgical techniques, storage, and instrumentation; the professionalization of eye banks through development of medical standards and accreditation programs; and the advent of laws and regulations leading to reimbursement changes and donor legislation. The author next identifies five strengths exhibited by the community to achieve those milestones and delineates crucial questions posed by an ever-expanding array of unprecedented demographic, socioeconomic, geopolitical, biological, regulatory, technological, cultural, and ethical challenges. CONCLUSIONS: Technological advances and collaborative efforts have brought the eye banking community to the enviable position it enjoys today. Only by building on its historical strengths can eye banking professionals worldwide successfully evolve their role in an increasingly complex future.
Article
The cell therapy industry is a fast-growing industry targeted toward a myriad of clinical indications. As the cell therapy industry matures and clinical trials hit their pivotal Phase 3 studies, there will be a significant need for scale-up, process validation, and critical raw material quality assurance. Part of the well discussed challenges of upscaling manufacturing processes there is a less discussed issue relating to the availability of raw materials in the needed quality and quantities. The FDA recently noted that over 80% of the 66 investigational new drug (IND) applications for mesenchymal stem cell (MSC) products analyzed described the use of FBS during manufacturing. Accumulated data from the past years show an acceleration in serum consumption by at least 10%-15% annually, which suggests that the global demand for serum may soon exceed the supply. Ongoing concerns of safety issues due to risks of various pathogen contaminations, as well as issues related to the aforementioned serum variability that can affect final product reproducibility, are strong motivators to search for serum substitutes or serum-free media. it is important to note that there are no accepted definitions for most of these terms which leads to misleading's and misunderstandings, where the same term might be defined differently by different vendors, manufacturer, and users. It is the drug developer's responsibility to clarify what the supplied labels mean and to identify the correct questions and audits to ensure quality. The paper reviews the available serum replacements, main components, basic strategies for replacement of serum and suggests definitions.
Article
A focused low-intensity pulsed ultrasound (FLIPUS) was used to investigate the effects of stimulation period, acoustic intensity and donor age on the osteogenic differentiation potential of rat mesenchymal stromal cells (rMSCs). rMSCs from 3- and 12-mo-old female Sprague Drawly rats were isolated from bone marrow and stimulated 20 min/d with either 11.7 or 44.5 mW/cm(2) (spatial average temporal average intensity) for 7 or 14 d. Osteogenic differentiation markers, i.e., Runt-related transcription factor 2 (RUNX2), osteocalcin (OCN) and degree of matrix calcification were analyzed. On day 7 of stimulation, OCN gene expression was enhanced 1.9-fold in cells from young rats when stimulated with low intensity. The low intensity also led to a 40% decrease in RUNX2 expression on day 7 in aged cells, whereas high intensity enhanced expression of RUNX2 on day 14. FLIPUS treatment with low intensity resulted in a 15% increase in extracellular matrix mineralization in young but not old rMSCs. These differences suggest the necessity of a donor-age related optimization of stimulation parameters.
Article
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